Anti-P-selectin antibodies

ABSTRACT

This invention relates to anti-P-selectin antibodies and, in particular, to anti-P-selectin antibodies and variants thereof that contain an Fc part derived from human origin and do not bind complement factor C1q. These antibodies have new and inventive properties causing a benefit for a patient suffering from critical limb ischemia or peripheral arterial occlusive disease (CLI/PAOD).

FIELD OF THE INVENTION

This invention relates generally to anti-P-selectin antibodies and, inparticular, to anti-P-selectin antibodies that do not bind complementfactor C1q. Preferably, these antibodies are human or humanizedantibodies.

BACKGROUND OF THE INVENTION

P-selectin (CD62P, GMP-140, PADGEM, LECAM-3) is a 140 kDacalcium-dependent carbohydrate-binding protein that is expressed on thesurfaces of activated platelets and endothelium in response to thrombinand other agonists (McEver et al., J Biol Chem 270:11025 (1995); Varki,Proc Natl Acad Sci USA 91:7390 (1994); Springer T A, Annu Rev Physiol57:827 (1995)). In both cell types, P-selectin is stored in secretorygranules, i.e. α-granules in platelets and Weibel-Palade bodies inendothelial cells (McEver et al., J Clin Invest 84:92 (1984)). It is atype I transmembrane glycoprotein which is composed of an NH₂-terminallectin domain, followed by an EGF-like domain, nine short consensusrepeats with homology to complement regulatory proteins, a transmembranedomain, and a short cytoplasmic tail (Johnston et al., Cell 56:1033(1989)). The structure of P-selectin is similar to the other two membersof the selectin family, E- and L-selectin, which are either expressed oncytokine-activated endothelial cells (E-selectin) or constitutivelyexpressed on most classes of leukocytes (L-selectin).

All selectins are known to bind with low affinity to small sialylated,fucosylated oligosaccharides such as sialyl Lewis x (sLe^(x); Foxall etal., J Cell Biol 117:895 (1992); Varki, Curr Opin Cell Biol 257:257(1992)). P— and L-selectin, but not E-selectin, also bind to particularsulfated carbohydrates, such as heparin sulfate (for review, see McEverand Cummings, J Clin Invest 100:S97 (1997)). High affinity ligands forP-selectin are mucin-like glycoproteins (McEver et al., J Biol Chem270:11025 (1995)), which consist of a polypeptide backbone with clustersof sialylated O-glycans. One sialomucin ligand to which P-selectin bindspreferentially is P-selectin Glycoprotein ligand-1 (PSGL-1, CD162),which is normally expressed as a homodimer with two disulfide-linkedsubunits with relative molecular masses of approximately 120 kDa bycirculating leukocytes. The binding site of P-selectin is localized tothe extreme NH₂-terminal part of PSGL-1. Through its binding to itsligands, P-selectin mediates rolling of the leukocytes on activatedplatelets and endothelial cells. The rolling process effectively reducesthe velocity of leukocyte movement, which is a prerequisite for firmadhesion and subsequent transmigration of leukocytes into thesubendothelium but also for the accumulation of leukocytes in thrombi.

Studies using P-selectin deficient mice and P-selectin-specific blockingantibodies have shown that P-selectin participates in thepathophysiology of numerous acute and chronic inflammatory diseasesincluding ischemia/reperfusion injury (Winn et al., J Clin Invest92:2042 (1993); Massberg et al., Blood 92:507 (1998)). In addition,there is a clear contribution of P-selectin in cardiovascular diseasesthat have an inflammatory component such as atherosclerosis (Collins etal., J Exp Med 191: 189 (2000); Johnson et al., J Clin Invest 99:1037(1997)), restenosis (Manka et al., Circulation 103:1000 (2001); Bienvenuet al., Circulation 103:1128 (2001)) and thrombosis (Kumar et al.,Circulation 99:1363 (1999); Andre et al., Proc Natl Acad Sci USA97:13835 (2000); Blann et al., Br. J. Haematol 108:191 (2000); Myers etal., Thromb Haemostasis 85: 423 (2001). Evidently, inhibition ofP-selectin function would be effective as a therapy in various diseasesinvolving leukocyte adherence to vascular endothelium or platelets (seee.g. WO 93/06863).

Antibodies against P-selectin have been described in the state of theart and investigated for their anti-inflammatory and anti-thromboticeffects. U.S. Pat. No. 4,783,399 and WO 93/06863 describe mousemonoclonal antibodies against P-selectin reactive with activatedplatelets. Geng J. G. et al (J. Biol. Chem., 266 (1991) 22313-22318)describe mouse monoclonal antibodies binding to P-selectin amino acid(aa) fragment aa 60-75 (Cys to Glu, counting according to Swiss-Protsequence P16109 which includes the signal sequence. WO 93/21956 refersto mouse monoclonal antibodies against P-selectin and humanizedantibodies of IgG1 subclass competing with a defined antibody, bindingin the presence of P-selectin fragment aa 60-75) and in the absence ofcalcium ions. None of the mentioned mouse monoclonal antibodies againsthuman P-selectin is useful for the treatment of human patients. Ahumanized antibody against P-selectin of human IgG1 subclass mentionedin WO 93/21956 is in pre-clinical development (www.mrctechnology.org).

SUMMARY OF THE INVENTION

The invention relates to antibodies characterized in that saidantibodies bind P-selectin and do not bind human complement factor C1q.Preferably the antibodies do also not bind to human Fcγ receptor on NKcells. The antibodies according to the invention contain a Fc partderived from human origin. Preferably these antibodies are humanized orhuman antibodies. The antibodies have new and inventive propertiescausing a benefit for a patient suffering from inflammatory andthrombotic disorders, especially from peripheral arterial occlusivedisease (PAOD) and critical limb ischemia (CLI).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that the antibodies of the invention inhibit the adhesionof leukocyte-like HL60 cells to purified P-selectin coated ontomicrotiter plates. The mutated antibodies are more potent than thenon-mutated parent antibody.

FIG. 2 shows the inhibitory activity of the antibodies of the inventionin the rosetting assay measuring the adhesion of thrombin-activatedplatelets to HL60 cells.

FIGS. 3 a and 3 b depicts the cross-reactivity of the antibodies of theinvention with rat and cynomologus P-selectin. FIG. 3 a: Theanti-P-selectin antibodies do not affect the adhesion ofthrombin-activated rat platelets to HL60 cells, whereas the commerciallyavailable polyclonal anti-P-selectin antibody (Pharmingen 09361A)inhibits this interaction. FIG. 3 b: The antibodies of the inventioninhibit the adhesion of activated cynomologus platelets to HL60 cells.

FIG. 4 a-c demonstrates the selectivity of the antibodies for P-selectinvs. E- and L-selectin by representative binding curves on P—, E- andL-selectin transfectants. The antibodies according to the invention bindto P-selectin CHO cells with EC₅₀ values in the range of 0.01 and 0.07μg/ml. EC₅₀ values on E-selectin CHO cells and L-selectin 300.19 cellsare preferably above 100 μg/ml.

FIG. 5 depicts the inhibitory activity of the antibodies of theinvention in a fully human flow system. They inhibit the adhesion ofhuman leukocytes to a platelet monolayer in a concentration-dependentmanner at a shear rate of 65/s.

FIG. 6 depicts the inhibitory effect of the antibodies of the inventionon the adhesion of leukocytes to human endothelial cells expressingP-selectin. FIG. 6 a demonstrates the total inhibition of leukocyteadhesion in % of the control, FIG. 6 b representatively shows theinhibitory effect of one of the antibodies on the absolute number of thedifferent leukocyte subsets.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to antibodies characterized in that saidantibodies bind P-selectin and do not bind human complement factor C1q.Preferably the antibodies do also not bind to human Fcγ receptor on NKcells. The antibodies according to the invention contain a Fc partderived from human origin. Preferably these antibodies are humanized orhuman antibodies. The antibodies have new and inventive propertiescausing a benefit for a patient suffering from inflammatory andthrombotic disorders, especially from peripheral arterial occlusivedisease (PAOD) and critical limb ischemia (CLI).

DEFINITIONS

The term “P-selectin” refers to a 140 kDa protein expressed by humanplatelets and endothelial cells, as described by Hsu-Lin et al., J BiolChem 259: 9121 (1984), and Mc Ever et al., J Clin Invest 84:92 (1989).This type I transmembrane glycoprotein is composed of an NH₂-terminallectin domain, followed by an epidermal growth factor (EGF)-like domainand nine consensus repeat domains. It is anchored in the membrane by asingle transmembrane domain and contains a small cytoplasmic tail. Thepresent invention provides antibodies, which are capable of inhibitingone or more of the biological activities mediated by P-selectin, forexample, its inflammatory or thrombotic activity. The antibodies bind toP-selectin and act by interfering with the binding of P-selectin to itsligand.

The term “P-selectin ligand” relates preferably to the high affinity andbiologically relevant ligand of P-selectin such as the mucin-likeglycoprotein P-selectin ligand glycoprotein-1 (PSGL-1), as described byMoore et al.; J Cell Biol 118:2445 (1992), Sako et al., Cell 75:1179(1993) PSGL-1 is a type I membrane protein with an extracellular domainrich in serines, threonines, and prolines, including a series ofdecameric repeats linked with clusters of sialylated O-glycans. It isnormally expressed as a homodimer with two disulfide-linked subunitswith relative molecular masses of approximately 120 kDa by circulatingleukocytes. The binding site of P-selectin is localized to the extremeNH₂-terminal part of PSGL-1. The sialomucin GPIbα which is expressed byplatelets and has structural similarities with PSGL-1 was recentlydemonstrated to be a platelet ligand for P-selectin (Romo et al., J ExpMed 190:803 (1999). The physiological consequences of GPIbα binding toP-selectin are still under investigation, the interaction, however, islikely to contribute to the rolling and adherence of platelets toactivated endothelial cells (Berndt et al., Thromb Haemost 86:178(2001). P-selectin also binds with low affinity to small sialated,fucosylated oligosaccharides such as sialyl Lewis x (Foxall et al., JCell Biol 117:895 (1992), Varki, Curr Opin Cell Biol 257 (1992) and toparticular sulfated carbohydrates, such as heparin sulfate (McEver etal., J Biol Chem 270:11025 (1995).

The term “antibody” encompasses the various forms of antibodies,preferably monoclonal antibodies including but not being limited towhole antibodies, antibody fragments, human antibodies, humanizedantibodies, chimeric antibodies and genetically engineered antibodies(variant or mutant antibodies) as long as the characteristic propertiesaccording to the invention are retained. Especially preferred are humanor humanized monoclonal antibodies, especially as recombinant humanantibodies.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of a singleamino acid composition.

The term “chimeric antibody” refers to a monoclonal antibody comprisinga variable region, i.e., binding region, from one source or species andat least a portion of a constant region derived from a different sourceor species, usually prepared by recombinant DNA techniques. Chimericantibodies comprising a murine variable region and a human constantregion are especially preferred. Such murine/human chimeric antibodiesare the product of expressed immunoglobulin genes comprising DNAsegments encoding murine immunoglobulin variable regions and DNAsegments encoding human immunoglobulin constant regions. Other forms of“chimeric antibodies” encompassed by the present invention are those inwhich the constant region has been modified or changed from that of theoriginal antibody to generate the properties according to the invention,especially in regard to C1q binding and/or Fc receptor (FcR) binding.Such “chimeric” antibodies are also referred to as “class-switchedantibodies.” Methods for producing chimeric antibodies involveconventional recombinant DNA and gene transfection techniques now wellknown in the art. See, e.g., Morrison, S. L., et al., Proc. Natl. Acad.Sci. USA 81 (1984) 6851-6855; U.S. Pat. Nos. 5,202,238 and 5,204,244.

The term “humanized antibody” refers to antibodies in which theframework or “complementarity determining regions” (CDR) have beenmodified to comprise the CDR of an immunoglobulin of differentspecificity as compared to that of the parent immunoglobulin. In apreferred embodiment, a murine CDR is grafted into the framework regionof a human antibody to prepare the “humanized antibody.” See, e.g.,Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S.,et al., Nature 314 (1985) 268-270. Particularly preferred CDRscorrespond to those representing sequences recognizing the antigensnoted above for chimeric and bifunctional antibodies. Other forms of“humanized antibodies” encompassed by the present invention are those inwhich the constant region has been modified or changed from that of theoriginal antibody to generate the properties according to the invention,especially in regard to C1q binding and/or Fc receptor (FcR) binding.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well-known in thestate of the art (van Dijk and van de Winkel, Curr Opin Pharmacol 5:368(2001). Human antibodies can also be produced in transgenic animals(e.g., mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. Transfer of the human germ-lineimmunoglobulin gene array in such germ-line mutant mice will result inthe production of human antibodies upon antigen challenge (see, e.g.,Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551-2555 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Yearin Immuno., 7:33 (1993)). Human antibodies can also be produced in phagedisplay libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1992);Marks et al., J. Mol. Biol, 222:581 (19991)). The techniques of Cole etal. and Boerner et al. are also available for the preparation of humanmonoclonal antibodies (Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol.,147(1):86-95 (1991)).As already mentioned for chimeric and humanizedantibodies according to the invention the term “human antibody” as usedherein also comprises such antibodies which are modified in the constantregion to generate the properties according to the invention, especiallyin regard to C1q binding and/or FcR binding. In addition the inventioncomprises human antibodies which bind to C1q and/or FcR. Such humanantibodies are characterized by a high selectivity for for P-selectinvs. E- and L-selectin. Such antibodies according to the invention bindto P-selectin expressing cells with EC₅₀ values in the range of 0.01 and0.07 μg/ml. EC₅₀ values on E-selectin and L-selectin expressing cellsare preferably above 100 μg/ml. Such antibodies are preferable useful asintermediates for manufacturing human antibodies with the propertiesaccording to the invention.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from a hostcell such as a NS0 or CHO cell or from an animal (e.g. a mouse) that istransgenic for human immunoglobulin genes or antibodies expressed usinga recombinant expression vector transfected into a host cell. Suchrecombinant human antibodies have variable and constant regions in arearranged form. The recombinant human antibodies according to theinvention have been subjected to in vivo somatic hypermutation. Thus,the amino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangerm line VH and VL sequences, may not naturally exist within the humanantibody germ line repertoire in vivo.

The “variable region” (variable region of a light chain (VL), variableregion of a heavy chain (VH)) as used herein denotes each of the pair oflight and heavy chains which is involved directly in binding theantibody to the antigen. The domains of variable human light and heavychains have the same general structure and each domain comprises fourframework (FR) regions whose sequences are widely conserved, connectedby three “hypervariable regions” (or complementarity determiningregions, CDRs). The framework regions adopt a β-sheet conformation andthe CDRs may form loops connecting the β-sheet structure. The CDRs ineach chain are held in their three-dimensional structure by theframework regions and form together with the CDRs from the other chainthe antigen binding site. The antibody heavy and light chain CDR3regions play a particularly important role in the bindingspecificity/affinity of the antibodies according to the invention andtherefore provide a further object of the invention.

The terms “hypervariable region” or “antigen-binding portion of anantibody” when used herein refer to the amino acid residues of anantibody which are responsible for antigen-binding. The hypervariableregion comprises amino acid residues from the “complementaritydetermining regions” or “CDRs”. “Framework” or “FR” regions are thosevariable domain regions other than the hypervariable region residues asherein defined. Therefore, the light and heavy chains of an antibodycomprise from N— to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. Especially, CDR3 of the heavy chain is the region whichcontributes most to antigen binding. CDR and FR regions are determinedaccording to the standard definition of Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)) and/or thoseresidues from a “hypervariable loop”.

The term “nucleic acid or nucleic acid molecule”, as used herein, isintended to include DNA molecules and RNA molecules. A nucleic acidmolecule may be single-stranded or double-stranded, but preferably isdouble-stranded DNA.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be dear fromthe context.

The “constant domains” are not involved directly in binding an antibodyto an antigen, but exhibit various effector functions. Depending on theamino acid sequence of the constant region of their heavy chains,antibodies or immunoglobulins are divided in the classes: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG1, IgG2, IgG3, and IgG4, IgA1 and IgA2. The heavychain constant regions that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ and μ, respectively. Theantibodies according to the invention are preferably of IgG type.

The Fc part of an antibody is directly involved in complementactivation, C1q binding and Fc receptor binding. While the influence ofan antibody on the complement system is dependent on certain conditions,binding to C1q is caused by defined binding sites in the Fc part. Suchbinding sites are known in the state of the art and described e.g. byBoakle et al., Nature 282 (1975) 742-743, Lukas et al., J. Immunol. 127(1981) 2555-2560, Brunhouse and Cebra, Mol. Immunol. 16 (1979) 907-917,Burton et al., Nature 288 (1980) 338-344, Thommesen et al., Mol.Immunol. 37 (2000) 995-1004, Idusogie et al., J. Immunol.164 (2000)4178-4184, Hezareh et al., J. Virology 75 (2001) 12161-12168, Morgan etal., Immunology 86 (1995) 319-324, EP 0307434. Such binding sites aree.g. L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numberingaccording to EU index of Kabat, see below). Antibodies of subclass IgG1,IgG2 and IgG3 usually show complement activation and C1q and C3 binding,whereas IgG4 do not activate the complement system and do not bind C1qand C3. As used herein the term “Fc part derived from human origin”denotes a Fc part which is either a Fc part of a human antibody of thesubclass IgG4 or a Fc part of a human antibody of the subclass IgG1,IgG2 or IgG3 which is modified in such a way that no C1q binding and/orFcR binding as defined below can be detected. A “Fc part of an antibody”is a term well known to the skilled artisan and defined on the basis ofpapain cleavage of antibodies. The antibodies according to the inventioncontain as Fc part a Fc part derived from human origin and preferablyall other parts of the human constant regions. Preferably the Fc part isa human Fc part and especially preferred either from human IgG4 subclassor a mutated Fc part from human IgG1 subclass. Mostly preferred are theFc parts and heavy chain constant regions shown in SEQ ID NO: 25-28 orof SEQ ID NO: 25 without PVA236 mutation.

Preferred Embodiments of the Invention

The invention comprises an antibody binding to P-selectin characterizedin that the variable heavy chain amino acid sequence CDR3 of saidantibody is selected from the group consisting of the heavy chain CDR3sequences SEQ ID NO: 38, 39, 40, 41 or 42.

The invention preferably provides an antibody binding to P-selectin,comprising a variable heavy chain and a variable light chain,characterized in that the variable heavy chain comprises CDR sequencesCDR1, CDR2 and CDR3 and CDR1 being selected from the group consisting ofSEQ ID NOs: 29, 30, 31, 32, CDR2 being selected from the groupconsisting of SEQ ID NOs: 33, 34, 35, 36, 37, CDR3 being selected fromthe group consisting of SEQ ID NOs: 38, 39, 40, 41, 42, wherein saidCDRs are selected independently of each other.

The antibody according to the invention is preferably characterized inthat the variable light chain comprises CDR sequences CDR1, CDR2 andCDR3, and CDR1 is selected from SEQ ID NOs: 43, 44, CDR2 is selectedfrom SEQ ID NOs: 45, 46 and CDR3 is selected from SEQ ID NOs: 47, 48,49, 50, 51, 52 wherein said CDRs are selected independently of eachother.

The antibody is preferably characterized in containing as heavy chainCDRs the CDRs of SEQ ID NO: 2 and as light chain CDRs the CDRs of SEQ IDNO: 1, as heavy chain CDRs the CDRs of SEQ ID NO: 4 and as light chainCDRs the CDRs of SEQ ID NO: 3, as heavy chain CDRs the CDRs of SEQ IDNO: 6 and as light chain CDRs the CDRs of SEQ ID NO: 5, as heavy chainCDRs the CDRs of SEQ ID NO: 8 and as light chain CDRs the CDRs of SEQ IDNO: 7, as heavy chain CDRs the CDRs of SEQ ID NO: 10 and as light chainCDRs the CDRs of SEQ ID NO: 9, as heavy chain CDRs the CDRs of SEQ IDNO: 12 and as light chain CDRs the CDRs of SEQ ID NO: 11, as heavy chainCDRs the CDRs of SEQ ID NO: 14 and as light chain CDRs the CDRs of SEQID NO: 13, as heavy chain CDRs the CDRs of SEQ ID NO: 16 and as lightchain CDRs the CDRs of SEQ ID NO: 15, as heavy chain CDRs the CDRs ofSEQ ID NO: 18 and as light chain CDRs the CDRs of SEQ ID NO: 17, asheavy chain CDRs the CDRs of SEQ ID NO: 20 and as light chain CDRs theCDRs of SEQ ID NO: 19, or as heavy chain CDRs the CDRs of SEQ ID NO: 22and as light chain CDRs the CDRs of SEQ ID NO: 21.

The CDR sequences can be determined according to the standard definitionof Kabat et al., Sequences of Proteins of Immunological Interest, 5thed., Public Health Service, National Institutes of Health, Bethesda, Md.(1991). CDRs on each chain are separated by framework amino acids. CDRsof SEQ ID NO: 1-22 are shown in SEQ ID NO: 29-52.

The antibody according to the invention is preferably characterized inthat said antibody binds P-selectin and comprises a variable heavy andlight region independently selected from the group consisting of

-   -   a) the heavy chain variable domain defined by amino acid        sequence SEQ ID NO:2 and the light chain variable domain defined        by SEQ ID NO:1;    -   b) the heavy chain variable domain defined by amino acid        sequence SEQ ID NO:4 and the light chain variable domain defined        by SEQ ID NO:3;    -   c) the heavy chain variable domain defined by amino acid        sequence SEQ ID NO:6 and the light chain variable domain defined        by SEQ ID NO:5;    -   d) the heavy chain variable domain defined by amino acid        sequence SEQ ID NO:8 and the light chain variable domain defined        by SEQ ID NO:7;    -   e) the heavy chain variable domain defined by amino acid        sequence SEQ ID NO:10 and the light chain variable domain        defined by SEQ ID NO:9;    -   f) the heavy chain variable domain defined by amino acid        sequence SEQ ID NO:12 and the light chain variable domain        defined by SEQ ID NO:11;    -   g) the heavy chain variable domain defined by amino acid        sequence SEQ ID NO:14 and the light chain variable domain        defined by SEQ ID NO:13;    -   h) the heavy chain variable domain defined by amino acid        sequence SEQ ID NO:16 and the light chain variable domain        defined by SEQ ID NO:15;    -   i) the heavy chain variable domain defined by amino acid        sequence SEQ ID NO:18 and the light chain variable domain        defined by SEQ ID NO:17;    -   j) the heavy chain variable domain defined by amino acid        sequence SEQ ID NO:20 and the light chain variable domain        defined by SEQ ID NO:19;    -   k) the heavy chain variable domain defined by amino acid        sequence SEQ ID NO:22 and the light chain variable domain        defined by SEQ ID NO:21.

The antibody according to the invention is preferably characterized inthat the heavy chain variable region comprises an amino acid sequenceindependently selected from the group consisting of SEQ ID NO: 2, 4, 6,8, 10, 12, 14, 16, 18, 20 and 22.

The antibody according to the invention is preferably characterized inthat the light chain variable region comprises an amino acid sequenceindependently selected from the group consisting of SEQ ID NO: 1, 3, 5,7, 9, 11, 13, 15, 17, 19 and 21.

The present invention refers to an antibody that binds P-selectin anddoes not bind complement factor C1q and/or Fc receptor. These antibodiesdo not elicit the complement dependent cytotoxicity (CDC) and/orantibody-dependent cellular cytotoxicity (ADCC). Preferably, thisantibody is characterized in that it binds P-selectin, contains a Fcpart derived from human origin and does not bind complement factor C1q.More preferably, this antibody is a human or humanized antibody.

The antibody according to the invention is preferably characterized inthat the constant chains are of human origin. Such constant chains arewell known in the state of the art and e.g. described by Kabat (see e.g.Johnson, G., and Wu, T. T., Nucleic Acids Res. 28 (2000) 214-218). Forexample a useful human heavy chain constant region comprises an aminoacid sequence independently selected from the group consisting of SEQ IDNO: 24, 25, 26, 27 and 28. For example an useful human light chainconstant region comprises an amino acid sequence of a kappa-light chainconstant region of SEQ ID NO: 23.

The effector functions mediated by the Fc part of the antibody Fc regionrefer to effector functions that operate after the binding of anantibody to an antigen (these functions involve the activation of thecomplement cascade and/or cell activation by a Fc receptor (FcR)).

The function of the complement cascade can be assessed by the CH50assay. Sheep red cells sensitized with anti-red cell antibodies (EA) areadded to test serum to activate the classical pathway resulting inhaemolysis. The volume of serum needed to lyse 50% of the red cellsdetermines the CH50 unit. The AP-CH50 measures the alternative and theterminal pathways. The procedure is similar except that rabbit red cellsare used. The alternative pathway is activated upon addition of testserum.

C1q and two serine proteases, C1r and C1s, form the complex C1, thefirst component of the complement dependent cytotoxicity (CDC) pathway.To activate the complement cascade C1 q binds to at least two moleculesof IgG1 or one molecule of IgM, attached to the antigenic target (Wardand Ghetie, Therapeutic Immunology 2:77-94 (1995)). Burton described(Molec. Immunol., 22(3):161-206 (1985)) that the heavy chain regioncomprising amino acid residues 318 to 337 is being involved incomplement fixation. Duncan and Winter (Nature 332:738-40 (1988)), usingsite directed mutagenesis, reported that Glu318, Lys320 and Lys322 formthe binding site to C1q. The role of Glu318, Lys320 and Lys 322 residuesin the binding of C1q was confirmed by the ability of a short syntheticpeptide containing these residues to inhibit complement mediated lysis.

The term “complement-dependent cytotoxicity (CDC)” refers to lysis ofP-selectin expressing human endothelial cells and platelets by theantibody according to the invention in the presence of complement. CDCis measured preferably by the treatment of P-selectin expressing humanendothelial cells and platelets with an antibody according to theinvention in the presence of complement. The cells are preferablylabeled with calcein. CDC is found if the antibody induces lysis of 20%or more of the target cells at a concentration of 30 μg/ml. However, theinventors have found that for the properties of the antibodies accordingto the invention reduced binding to the complement factor C1q in anELISA assay is essential. In such an assay in principle an ELISA plateis coated with concentration ranges of the antibody, to which purifiedhuman C1q or human serum is added. C1q binding is detected by anantibody directed against C1q followed by a peroxidase-labeledconjugate. Detection of binding (maximal binding Bmax) is measured asoptical density at 405 nm (OD405) for peroxidase substrate ABTS(2,2′-Azino-di-[3-ethylbenzthiazoline-6-sulfonate (6)]. Accordingly thepresent invention refers to an antibody, characterized in thatnon-binding of the antibody to complement factor C1q refers to such anELISA assay measurement wherein the maximal binding (Bmax) of C1q to theantibody at a concentration of 10 μg/ml of the antibody is ≦30% of Bmaxof the antibody LC 1004-002 of cell line hu-Mab<P-selectin>LC 1004-002(DSM ACC2641).preferably 20% or lower.

It is further preferred, that an antibody according to the inventionshows a reduced binding to complement factor C3 in an ELISA assay. Theassay is performed in the same manner as the C1q assay. In such an assayin principle an ELISA plate is coated with concentration ranges of theantibody, to which purified human C3 or human serum is added. C3 bindingis detected by an antibody directed against C3 followed by aperoxidase-labeled conjugate. Detection of binding (maximal bindingBmax) is measured as optical density at 405 nm (OD405) for peroxidasesubstrate ABTS (2,2′-Azino-di-[3-ethylbenzthiazolinesulfonate (6)].Accordingly the present invention refers to an antibody, characterizedin that non-binding of the antibody to complement factor C3 refers tosuch an ELISA assay measurement wherein the maximal binding (Bmax) of C3to the antibody at a concentration of 10 μg/ml of the antibody is 10% ofBmax of antibody LC 1004-002 of cell line hu-Mab<P-selectin>LC 1004-002(DSM ACC2641)., preferably 5% or lower.

The term “antibody-dependent cellular cytotoxicity (ADCC)” is a functionmediated by Fc receptor binding and refers to lysis of P-selectinexpressing target cells by an antibody according to the invention in thepresence of effector cells. ADCC is measured preferably by the treatmentof a preparation of P-selectin expressing endothelial cells with anantibody according to the invention in the presence of effector cellssuch as freshly isolated PBMC (peripheral blood mononuclear cells) orpurified effector cells from buffy coats, like monocytes or NK (naturalkiller) cells. Target cells are labeled with ⁵¹Cr and subsequentlyincubated with the antibodies. The labeled cells are incubated witheffector cells and the supernatant is analyzed for released ⁵¹Cr.Controls include the incubation of the target endothelial cells witheffector cells but without the antibody. The capacity of the antibodiesto induce the initial steps mediating ADCC was determined by measuringtheir binding to Fcγ receptors expressing cells, such as granulocytes(expressing FcγRII and RIII), NK cells (expressing FcγRIII) andmonocytes (expressing FcγRI and RII).

Fc receptor binding effector functions can be mediated by theinteraction of the Fc region of an antibody with Fc receptors (FcRs),which are specialized cell surface receptors on hematopoietic cells. Fcreceptors belong to the immunoglobulin superfamily, and have been shownto mediate both the removal of antibody-coated pathogens by phagocytosisof immune complexes, and the lysis of erythrocytes and various othercellular targets (e.g. tumor cells) coated with the correspondingantibody, via antibody dependent cell mediated cytotoxicity (ADCC). Vande Winkel and Anderson, J. Leuk. Biol. 49:511-24 (1991). FcRs aredefined by their specificity for immunoglobulin isotypes; Fc receptorsfor IgG antibodies are referred to as FcγR, for IgE as FcεR, for IgA asFcαR and so on. Fc receptor binding is described e.g. in Ravetch andKinet, Ann. Rev. Immunol. 9 (1991) 457-492, Capel et al., Immunomethods4 (1994) 32-34, de Haas et al., J. Lab. Clin. Med. 126 (1995) 330-341and Gessner et al., Ann. Hematol. 76 1998) 231-248. The antibodiesaccording to the invention preferably show a reduced binding to Fcγreceptors, preferably to FγcRI, -IIA, -IIB, and/or IIIA.

The antibodies according to the present invention antibodies preferablydo not elicit any effector function and do not bind to FcγR presented onNK cells. The term “no binding of FcγR” therefore means that in anantibody concentration of 10 μg/ml the binding of an antibody accordingto the invention to NK cells is 1% or less of the binding found forantibody LC 1004-002 of cell line hu-Mab<P-selectin>LC 1004-002 (DSMACC2641).

While IgG4 shows reduced FcR binding, antibodies of other IgG subclassesshow strong binding. However Pro238, Asp265, Asp270, Asn297 (loss of Fccarbohydrate), Pro329 and 234, 235, 236 and 237 Ile253, Ser254, Lys288,Thr307, Gln311, Asn434, and His435 are residues which provides ifaltered also reduced FcR binding (Shields et al. J. Biol. Chem. 276(2001), 6591-6604, Lund et al. FASEB J. 9 (1995), 115-119, Morgan et al.Immunology 86 (1995) 319-324, EP 0307434 ). Preferably an antibodyaccording to the invention is in regard to FcR binding of IgG4 subclassor of IgG1 or IgG2 subclass with a mutation in S228, L234, L235 and/orD265, and/or contains the PVA236 or GLPSS331 mutation. Especiallypreferred are the mutations S228P (IgG4), L234A (IgG1), L235A (IgG1),L235E (IgG4), GLPSS331(IgG1) and/or PVA236 (IgG1). Preferredcombinations of mutations are also shown in table 1. An additionalpreferred combination is D265A/N297A.

The term “binding to P-selectin” as used herein means the binding of theantibody to P-selectin in either a BIAcore assay (Pharmacia BiosensorAB, Uppsala, Sweden) or in an ELISA in which either purified P-selectinor P-selectin CHO transfectants are coated onto microtiter plates.

In the BIAcore assay the antibody is bound to a surface and binding ofP-selectin is measured by Surface Plasmon Resonance (SPR). The affinityof the binding is defined by the terms ka (rate constant for theassociation of the antibody from the antibody/antigen complex), kd(dissociation constant), and K_(D) (kd/ka). The antibodies according tothe invention show a K_(D) of 10⁻⁸ or less, preferably of about 10⁻¹¹ to10⁻⁹ M (see examples). Accordingly, the present invention refers to anantibody as decribed above, wherein the antibody bind to P-selectin witha K_(D) value of less than 10⁻⁸ M in a BIAcore assay, preferably whereinthe K_(D) range is 10⁻¹¹ to 10⁻⁹ M.

Preferably, the antibody is of IgG1 or IgG4 human subtype. Morepreferably, the antibody is characterized in that the antibody is anantibody of human subclass IgG1, containing (comprising) at least onemutation in L234, L235, D270, N297, E318, K320, K322, P331 and/or P329or an antibody of human subclass IgG4, containing (comprising) at leastone mutation in L235 and S228 (numbering according to EU index).

In the P-selectin-specific ELISA purified P-selectin is coated ontomicrotiter plates and the binding of the antibody to P-selectin isdetected with a biotinylated anti-human IgG and the usual steps of anELISA. The EC₅₀ values in this assay range preferably between 0.002 and0.03 μg/ml on P-selectin CHO cells, i.e. the present invention refers toantibodies, wherein the EC50 values for P-selectin binding are in therange of 0.002 to 0.03 μg/ml on P-selectin presenting CHO cells in anELISA assay. In an assay in which P-selectin expressing CHOtransfectants are coated onto the microtiter plate, the EC50 valuesrange between 0.01 and 0.08 μg/ml, preferably between 0.01 and 0.04μg/ml.

EC₅₀ values on E- and L-selectin transfectants are preferably above 100μg/ml. The antibodies of the present invention are characterized in thatthey bind at least 1000 fold more specifically to P-selectin than to E-and/or L-selectin as measured by EC₅₀ values in an ELISA assay, whereinP— and E- and/or L-selectin are coated onto the microtiter plate.

The term “inhibiting the binding of the P-selectin ligand to P-selectin”as used herein refers to the binding of purified or cell-expressedP-selectin to its ligand presented on HL60 cells. The binding ofP-selectin to its ligand is inhibited by the antibodies according to theinvention. The inhibition is measured as IC₅₀ in in vitro assaysanalyzing the capacity of the antibody to inhibit binding of P-selectinto a ligand. Such assays are described in the Examples. They use assuitable sources of P-selectin affinity purified P-selectin andactivated platelets and as suitable sources of the ligand leukocyte-likecells, such as HL60 cells. In such assays the adhesion of HL60 cells,expressing PSGL-1 as the physiologically relevant ligand of P-selectin,to P-selectin or activated platelets is measured without and withincreasing concentrations of the antibody. The IC₅₀ values are measuredas average values of at least three independent measurements. Inhibitingmeans an IC₅₀ value of no more than 1 μg/ml, preferably 0.5 to 0.08μg/ml.

The antibodies of the present invention inhibit the adhesion ofleukocyte-like HL60 cells to purified P-selectin with IC50 values in therange of 0.08 to 0.5 μg/ml, preferably 0.08 to −0.11 μg/ml. The adhesionof leukocyte-like HL60 cells to activated platelets is inhibited withIC50 values in the range of 0.05 to 0.3 μg/ml.

Accordingly, further embodiments of the present invention refer toantibodies, characterized in that the EC50 values for P-selectin bindingis in the range of 0.01 to 0.08 μg/ml in an ELISA assay whereinP-selectin expressing CHO transfectants are coated onto the microtiterplate. The preferred range is 0.01 to 0.04 μg/ml. The EC50 values on E-and L-selectin transfectants are above 100 μg/ml. In a furtherembodiment the antibodies of the present invention inhibit the adhesionof leukocyte-like HL60 cells to purified P-selelctin with IC50 valuesbetween 0.08 to 0.5 μg/ml. The preferred range is 0.08 to 0.11 μg/ml.

The antibodies of the present invention inhibit the interaction ofleukocytes with a monolayer of platelets by preferably more than 70% ina fully human flow system (at a concentration of 10 μg/ml). In additionthese antibodies inhibit the adhesion of leukocytes to activatedendothelial cells in a human flow system in the range of 60-90% at aconcentration of 3 μg/ml (with differential effects on leukocytesubtypes).

The antibodies of the present invention are preferably capable ofbinding to P-selectin in the presence of the P-selectin fragment aa60-75 (Swiss-Prot sequence P16109) and/or do not competitively inhibitthe binding of an antibody secreted by a cell line designated ATCCAccession No. HB11041 to P-selectin.

The antibodies of the invention preferably do not inhibit theinteraction of P-selectin with platelet membrane glycoprotein GPIbα inan ELISA assay format. In the ELISA glycocalicin, the solubleextracellular portion of GPIbα was immobilized on the wells ofmicrotiter plates, as described (Romo et al., J Exp Med 190:803 (1999),and the binding of purified P-selectin after preincubation with theP-selectin HuMabs was detected with a polyclonal anti-P-selectinantibody.

In a further preferred embodiment of the present invention, theantibody, characterized in that does not bind the C3 protein, morepreferably it is characterized in that it does not elicitcomplement-dependent cytotoxicity (CDC). Further, the antibody may becharacterized it does not bind to Fcγ receptors on NK effector cells.Preferably, the antibody is characterized that it is an antibody ofhuman subclass IgG1, containing at least one mutation in L234, L235,D270, N297, E318, K320, K322, P331 and/or P329 or an antibody of humansubclass IgG4, containing at least one mutation in L235 and S228(numbering according to EU index). In a further preferred embodiment,the antibody is characterized in that it does not elicitantibody-dependent cellular cytotoxicity (ADCC).

In an even more preferred embodiment, the antibodies of the presentinvention are characterized in that they bind P-selectin and that theycomprise a variable region independently selected from the groupconsisting of

-   -   a) the light chain variable domain defined by amino acid        sequence SEQ ID NO:1 and the heavy chain variable domain defined        by SEQ ID NO:2;    -   b) the light chain variable domain defined by amino acid        sequence SEQ ID NO:3 and the heavy chain variable domain defined        by SEQ ID NO:4;    -   c) the light chain variable domain defined by amino acid        sequence SEQ ID NO:5 and the heavy chain variable domain defined        by SEQ ID NO:6;    -   d) the light chain variable domain defined by amino acid        sequence SEQ ID NO:7 and the heavy chain variable domain defined        by SEQ ID NO:8;    -   e) the light chain variable domain defined by amino acid        sequence SEQ ID NO:9 and the heavy chain variable domain defined        by SEQ ID NO:10;    -   f) the light chain variable domain defined by amino acid        sequence SEQ ID NO:11 and the heavy chain variable domain        defined by SEQ ID NO:12;    -   g) the light chain variable domain defined by amino acid        sequence SEQ ID NO:13 and the heavy chain variable domain        defined by SEQ ID NO:14;    -   h) the light chain variable domain defined by amino acid        sequence SEQ ID NO:15 and the heavy chain variable domain        defined by SEQ ID NO:16;    -   i) the light chain variable domain defined by amino acid        sequence SEQ ID NO:17 and the heavy chain variable domain        defined by SEQ ID NO:18;    -   j) the light chain variable domain defined by amino acid        sequence SEQ ID NO:19 and the heavy chain variable domain        defined by SEQ ID NO:20; and    -   k) the light chain variable domain defined by amino acid        sequence SEQ ID NO:21 and the heavy chain variable domain        defined by SEQ ID NO:22.

Preferably, the antibodies comprise the light chain variable domaindefined by amino acid sequence SEQ ID NO:3 and the heavy chain variabledomain defined by SEQ ID NO:4.

The preferred antibodies are characterized in that the antibodies are ofhuman IgG4 subclass or comprise at least one amino acid mutation causingnon-binding to complement factor C1q. These variant antibodies comprisefor example the amino acid sequence independently selected from thegroup consisting of SEQ ID NO: 25 or SEQ ID NO:26 and SEQ ID NO:28.

A “variant” anti-P-selectin antibody, refers herein to a molecule whichdiffers in amino acid sequence from a “parent” anti-P-selectin antibodyamino acid sequence by virtue of addition, deletion and/or substitutionof one or more amino acid residue(s) in the parent antibody sequence. Inthe preferred embodiment, the variant comprises one or more amino acidsubstitution(s) in one or more constant or variable region(s) of theparent antibody, preferably in the constant region. For example, thevariant may comprise at least one, e.g. from about one to about ten, andpreferably from about two to about five, substitutions in one or morevariable regions of the parent antibody. Ordinarily, the variant willhave an amino acid sequence having at least 90% amino acid sequenceidentity with the parent antibody constant and/or variable domainsequences, more preferably at least 95%, and most preferably at least99%.

Identity or homology with respect to this sequence is defined herein asthe percentage of amino acid residues in the candidate sequence that areidentical with the parent antibody residues, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. None of N-terminal, C-terminal, or internalextensions, deletions, or insertions into the antibody sequence shall beconstrued as affecting sequence identity or homology. The variantretains the ability to bind human P-selectin and preferably hasproperties, which are superior to those of the parent antibody. Forexample, the variant may have a stronger binding affinity, enhancedability to treat a disease associated with critical limb ischemia orperipheral arterial occlusive disease (CLI/PAOD).

The variant antibody of particular interest herein is one which displaysat least about 4 fold, enhancement in inhibitory activity in theadhesion assay when compared to the parent antibody because of theelimination of the binding to the Fcγ receptors.

The “parent” antibody herein is one, which is encoded by an amino acidsequence used for the preparation of the variant. Preferably, the parentantibody has a human framework region and, if present, has humanantibody constant region(s). For example, the parent antibody may be ahumanized or human antibody.

The antibodies according to the invention include, in addition, suchantibodies having “conservative sequence modifications”, nucleotide andamino acid sequence modifications, which do not affect or alter theabove-mentioned characteristics of the antibody according to theinvention. Modifications can be introduced by standard techniques knownin the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions include ones in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a human anti-P-selectinantibody can be preferably replaced with another amino acid residue fromthe same side chain family.

Amino acid substitutions can be performed by mutagenesis based uponmolecular modeling as described by Riechmann, L., et al., Nature 332(1988) 323-327 and Queen, C., et al., Proc. Natl. Acad. Sci. USA 86(1989) 10029-10033.

In a further preferred embodiment the antibodies comprise an κ-lightchain constant region as defined by SEQ ID NO:23.

Preferred antibodies according to the invention are antibodies definedas IgG1v1 (PVA-236; GLPSS331 as specified by E233P; L234V; L235A; deltaG236; A327G; A330S; P331S), IgG1v2 (L234A; L235A) and IgG4v1 (S228P;L235E).

In a further preferred embodiment, these antibodies also compriseantibody fragments selected from the group consisting of Fab, F(ab′)₂and single-chain fragments.

The invention further comprises a method for the production of anantibody according to the invention comprising the steps of a)transforming a host cell with a first nucleic acid sequence encoding alight chain of a parent human antibody according to the invention and asecond DNA sequence encoding a heavy chain of said parent human antibodywherein the Fc part is modified in that said Fc part does not bindcomplement factor C1q and/or Fc receptor; b) expressing said first andsecond DNA sequence so that said antibody heavy and light chains areproduced and c) recovering said antibody from the host cell or host cellculture.

The invention also refers to intermediate antibodies, i.e.anti-P-selectin antibodies characterized in that these antibodies arehuman or humanized antibodies and bind at least 1000 fold morespecifically to P-selectin than to E- or L-selectin as measured in anELISA assay wherein P— and E- and/or L-selectin are coated onto themicrotiter plate. Preferably these antibodies are IgG1 or IgG4antibodies. These antibodies may also comprise the amino acid sequenceas defined by SEQ ID NO:24 γ1 heavy chain constant region or SEQ IDNO:27 γ4 heavy chain constant region. Especially, these antibodies referto the antibodies produced by a cell line selected from the groupconsisting of hu-Mab<P-selectin>LC 1004-001 (DSM ACC2640),hu-Mab<P-selectin>LC 1004-002 (DSM ACC2641) and hu-Mab<P-selectin>LC1004-017(DSM ACC2642).

The antibodies according to the invention include, in addition, suchantibodies having “conservative sequence modifications”, nucleotide andamino acid sequence modifications, which do not affect or alter theabove-mentioned characteristics of the antibody according to theinvention. Modifications can be introduced by standard techniques knownin the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions include ones in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a human anti-P-selectinantibody can be preferably replaced with another amino acid residue fromthe same side chain family.

Amino acid substitutions can be performed by mutagenesis based uponmolecular modeling as described by Riechmann, L., et al., Nature 332(1988) 323-327 and Queen, C., et al., Proc. Natl. Acad. Sci. USA 86(1989) 10029-10033.

The invention further embodies an antibody (such as for example anantibody molecule) containing a Fc part derived from human originwherein the antibody binds to P-selectin and is non-binding tocomplement factor C1q and wherein, in a further embodiment, saidantibody is selected from the group consisting of a) human subclass IgG1antibody comprising at least one mutation in L234, L235, D270,N297,E318, K320, K322, P331 and P329 and b) human subclass IgG4 antibodywherein S228 is replaced by P and L235 is replaced by E. In anotherfurther embodiment, the antibody is an anti-P-selectin antibody producedby a hybridoma cell line selected from the group consisting of DSMACC2640, DSM ACC2641 and DSM ACC2642. The invention also disclosespharmaceutical compositions of the antibody of the invention, furthercomprising at least one pharmaceutically acceptable excipient.

The invention further comprises a method for the preparation of such anantibody according to the invention under conditions which allowsynthesis and recovery of the antibody, as well as pharmaceuticalcomposistions of the such produced antibody. The method of preparationmay additionally comprise a nucleic acid molecule which encodes theantibody of the invention, a vector that comprises said nucleic acidmolecule, and/or a host cell comprising said vector. The invention alsocomprises a kit for the detection of the presence of P-selectin proteincomprising the said antibody of the invention as disclosed above andfurther alternatively comprises one or more of the group consisting of anucleic acid molecule which encodes the said antibody of the invention,a vector that comprises said nucleic acid molecule, and/or a host cellcomprising said vector.

The invention also further comprises a method for treatment of a patientin need of therapy comprising administering to the patient atherapeutically effective amount of the antibody of the invention. Theinvention further embodies a medicament comprising the antibody of theinvention for use in such therapy administration and treatment and inparticular for the treatment of inflammatory and thrombotic disorders,more particularly for the treatment of PAOD and CLI.

The present invention also comprises nucleic acid molecules encoding anantibody mentioned above, the corresponding vectors comprising thesenucleic acids and the corresponding host cell for these vectors. Theinvention encompasses a method for the preparation of the antibodiescomprising culturing the corresponding host cells under conditions thatallow synthesis of said antibody molecules and recovering saidantibodies from said culture, e.g. by expressing a nucleic acid encodinga heavy chain and a nucleic acid encoding a light chain in a prokaryoticor eukaryotic host cell and recovering said polypeptide from said cell

Diagnostic and therapeutic uses for the antibody are contemplated. Inone diagnostic application, the invention provides a method fordetermining the presence of the P-selectin protein comprising exposing asample suspected of containing P-selectin to the anti-P-selectinantibody and determining binding of the antibody to the sample. For thisuse, the invention provides a kit comprising the antibody andinstructions for using the antibody to detect the P-selectin protein.

The antibodies of the present invention are useful for treatment ofinflammatory and thrombotic diseases. Such diseases include vasculardisorders such as atherosclerosis, arterial and deep venous thrombosis,restenosis after angioplasty or stent placement. Preferred applicationsare peripheral arterial occlusive disease (PAOD) and critical limbischemia (CLI). Other applications are the treatment of post-ischemicleukocyte-mediated tissue damage caused by myocardial infarction,cerebral ischemic event (e.g. stroke), renal infarction, and the like.The antibodies are also suitable for treatment of sepsis, acuteleukocyte-mediated lung-injury, and allergic reactions such as asthma.Other applications are the prevention of organ transplant rejection andautoimmune diseases including rheumatoid arthritis. In addition, tumormetastasis can be prevented by inhibiting the adhesion of circulatingcancer cells.

The invention further provides a method for treating a mammal sufferingfrom the abovementioned inflammatory and thrombotic disorders,especially from PAOD and CLI (peripheral arterial occlusive disease orcritical limb ischemia).

The invention further provides the use of the above antibodies fortherapy, e.g. for the manufacture of medicaments for the treatment ofthese diseases.

The invention relates also to the use of the antibodies as defined abovefor the manufacture of a pharmaceutical composition and comprises apharmaceutical composition containing an antibody according to theinvention with a pharmaceutically effective amount, optionally togetherwith a buffer and/or an adjuvant useful for the formulation ofantibodies for pharmaceutical purposes.

The invention further provides pharmaceutical compositions comprisingsuch antibodies in a pharmaceutically acceptable carrier. In oneembodiment, the pharmaceutical composition may be included in an articleof manufacture or kit.

The invention further provides hybridoma cell lines, which produce suchantagonistic monoclonal antibodies, e.g. the parent antibodies,according to the invention.

The preferred hybridoma cell lines according to the invention,hu-Mab<P-selectin>LC 1004-001 (antibody HuMab 001) hu-Mab<P-selectin>LC1004-002 (antibody HuMab 002) and hu-Mab<P-selectin>LC 1004-017(antibody HuMab 017) were deposited, under the Budapest Treaty on theinternational recognition of the deposit of microorganisms for thepurposes of patent procedure, with Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH (DSMZ), Germany: Cell line Deposition No. Date ofDeposit hu-Mab<P-selectin>LC 1004-001 DSM ACC2640 30.03.2004hu-Mab<P-selectin>LC 1004-002 DSM ACC2641 30.03.2004hu-Mab<P-selectin>LC 1004-017 DSM ACC2642 30.03.2004

The antibodies obtainable from said cell lines are preferred embodimentsof the invention.

The antibodies according to the invention are preferably produced byrecombinant means. Such methods are widely known in the state of the artand comprise protein expression in prokaryotic and eukaryotic cells withsubsequent isolation of the antibody polypeptide and usuallypurification to a pharmaceutically acceptable purity. For the proteinexpression, nucleic acids encoding light and heavy chains or fragmentsthereof are inserted into expression vectors by standard methods.Expression is performed in appropriate prokaryotic or eukaryotic hostcells like CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells,yeast, or E.coli cells, and the antibody is recovered from the cells(supernatant or cells after lysis).

Recombinant production of antibodies is well-known in the state of theart and described, for example, in the review articles of Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., ProteinExpr. Purif. 8 (1996) 271-282; Kaufman, R. J., Mol. Biotechnol. 16(2000) 151-161; Werner, R. G., Drug Res. 48 (1998) 870-880.

The antibodies may be present in whole cells, in a cell lysate, or in apartially purified or substantially pure form. Purification is performedin order to eliminate other cellular components or other contaminants,e.g. other cellular nucleic acids or proteins, by standard techniques,including alkaline/SDS treatment, column chromatography and others wellknown in the art. See Ausubel, F., et al., ed. Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York(1987).

Expression in NS0 cells is described by, e.g., Barnes, L. M., et al.,Cytotechnology 32 (2000) 109-123; and Barnes, L. M., et al., Biotech.Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g.,Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning ofvariable domains is described by Orlandi, R., et al., Proc. Natl. Acad.Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci.USA 89 (1992) 4285-4289; and Norderhaug, L., et al., J. Immunol. Methods204 (1997) 77-87. A preferred transient expression system (HEK 293) isdescribed by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30(1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)191-199.

The control sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters, enhancersand polyadenylation signals.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading frame. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The monoclonal antibodies are suitably separated from the culture mediumby conventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. DNA and RNAencoding the monoclonal antibodies are readily isolated and sequencedusing conventional procedures. The hybridoma cells can serve as a sourceof such DNA and RNA. Once isolated, the DNA may be inserted intoexpression vectors, which are then transfected into host cells such asHEK 293 cells, CHO cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of recombinantmonoclonal antibodies in the host cells.

Amino acid sequence variants (or mutants) of a human P-selectin antibodyare prepared by introducing appropriate nucleotide changes into theantibody DNA, or by nucleotide synthesis. Such modifications can beperformed, however, only in a very limited range, e.g. as describedabove. For example, the modifications do not alter the abovementionedantibody characteristics such as the IgG isotype and epitope binding,but may improve the yield of the recombinant production, proteinstability or facilitate the purification.

Any cysteine residue not involved in maintaining the proper conformationof the anti-P-selectin antibody also may be substituted, generally withserine, to improve the oxidative stability of the molecule and preventaberrant crosslinking. Conversely, cysteine bond(s) may be added to theantibody to improve its stability (particularly where the antibody is anantibody fragment such as an Fv fragment).

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.Glycosylation of antibodies is typically N-linked. N-linked refers tothe attachment of the carbohydrate moiety to the side chain of anasparagine residue. The tripeptide sequences asparagine-X-serine andasparagine-X-threonine, where X is any amino acid except proline, arethe recognition sequences for enzymatic attachment of the carbohydratemoiety to the asparagine side chain. Thus, the presence of either ofthese tripeptide sequences in a polypeptide creates a potentialglycosylation site. Addition of glycosylation sites to the antibody isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tripeptide sequences (forN-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants ofanti-P-selectin antibodies are prepared by a variety of methods known inthe art. These methods include, but are not limited to, isolation from anatural source (in the case of naturally occurring amino acid sequencevariants) or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of humanized anti-P-selectinantibody.

The invention also pertains to immunoconjugates comprising the antibodyaccording to the invention conjugated to a cytotoxic agent such as achemotherapeutic agent, toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant or animal origin, or fragments thereof), aradioactive isotope (i.e., a radioconjugate) or a prodrug of an agentfor the prophylaxis or treatment of inflammatory and thromboticdisorders, especially from PAOD and CLI. Conjugates of the antibody andcytotoxic agent are made using a variety of bifunctional proteincoupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate(SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters;(such as dimethyl adipimidate HCL), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azidocompounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediatnine),diisocyanates (such as tolyene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Forexample, a ricin immunotoxin can be prepared as described in Vitetta, E.S., et al., Science 238 (1987) 1098-1104). Carbon-14-labeled1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid(MX-DTPA) is an exemplary chelating agent for conjugation ofradionucleotide to the antibody. See WO 94/11026.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the antibody. These procedures areadvantageous in that they do not require production of the antibody in ahost cell that has glycosylation capabilities for N— or O-linkedglycosylation. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO 87/05330, and in Aplin, J. D., and Wriston, J. C. Jr.,CRC Crit. Rev. Biochem. (1981) 259-306.

Removal of any carbohydrate moieties present on the antibody may beaccomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the antibody to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theantibody intact. Chemical deglycosylation is described by Sojahr, H. T.,and Bahl, O. P., Arch. Biochem. Biophys. 259 (1987) 52-57 and by Edge,A. S., et al. Anal. Biochem. 118 (1981) 131-137. Enzymatic cleavage ofcarbohydrate moieties on antibodies can be achieved by the use of avariety of endo- and exo-glycosidases as described by Thotakura, N. R.,and Bahl, O. P., Meth. Enzymol. 138 (1987) 350-359.

Another type of covalent modification of the antibody comprises linkingthe antibody to one of a variety of nonproteinaceous polymers, eg.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

In yet another aspect, the invention provides isolated B-cells from atransgenic non-human animal, e.g. a transgenic mouse, which express thehuman anti-P-selectin antibodies (e.g. the parent antibodies produced bya cell line selected from the group consisting of hu-Mab<P-selectin>LC1004-001 (DSM ACC2640), hu-Mab<P-selectin>LC 1004-002 (DSM ACC2641) andhu-Mab<P-selectin>LC 1004-017(DSM ACC2642) according to the invention.KM mice are suitable transchromosomal mice. The KM mouse contains ahuman heavy chain transchromosome and a human kappa light chaintransgene. The endogenous mouse heavy and light chain genes also havebeen disrupted in the KM mice such that immunization of the mice leadsto production of human immunoglobulins rather than mouseimmunoglobulins. Construction of KM mice and their use to raise humanimmunoglobulins is described in detail in WO 02/43478.

Preferably, the isolated B cells are obtained from a transgenicnon-human animal, e.g., a transgenic mouse, which has been immunizedwith a purified or recombinant form of P-selectin antigen and/or cellsexpressing P-selectin. Preferably, the transgenic non-human animal, e.g.a transgenic mouse, has a genome comprising a human heavy chaintransgene and a human light chain transgene encoding all or a portion ofan antibody of the invention. The isolated B-cells are then immortalizedto provide a source (e.g. a hybridoma) of human anti-P-selectinantibodies. Accordingly, the present invention also provides a hybridomacapable of producing human monoclonal antibodies according to theinvention. In one embodiment, the hybridoma includes a B cell obtainedfrom a transgenic non-human animal, e.g., a transgenic mouse having agenome comprising a human heavy chain transgene and a human light chaintransgene encoding all or a portion of an antibody of the invention,fused to an immortalized cell.

In a particular embodiment, the transgenic non-human animal is atransgenic mouse having a genome comprising a human heavy chaintransgene and a human light chain transgene encoding all or a portion ofan antibody of the invention. The transgenic non-human animal can beimmunized with a purified or enriched preparation of P-selectin antigenand/or cells expressing P-selectin. Preferably, the transgenic non-humananimal, e.g. the transgenic mouse, is capable of producing P-selectinisotypes of human monoclonal antibodies to P-selectin.

The human monoclonal antibodies according to the invention can beproduced by immunizing a transgenic non-human animal, e.g. a transgenicmouse, having a genome comprising a human heavy chain transgene and ahuman light chain transgene encoding all or a portion of an antibody ofthe invention, with a purified or enriched preparation of P-selectinantigen and/or cells expressing P-selectin. B cells (e.g. splenic Bcells) of the animal are then obtained and fused with myeloma cells toform immortal, hybridoma cells that secrete human monoclonal antibodiesagainst P-selectin.

In a preferred embodiment, human monoclonal antibodies directed againstP-selectin can be generated using transgenic mice carrying parts of thehuman immune system rather than the mouse system. These transgenic mice,referred to herein as “HuMab” mice, contain a human immunoglobulin geneminiloci that encodes unrearranged human immunoglobulin genes whichinclude the heavy (μ and γ) and κ light chain (constant region genes),together with targeted mutations that inactivate the endogenous μ and κchain loci (Lonberg, N., et al., Nature 368 (1994) 856-859).Accordingly, the mice exhibit reduced expression of mouse IgM or K, andin response to immunization, the introduced human heavy and light chaintransgenes undergo class switching and somatic mutation to generate highaffinity human IgG monoclonal antibodies (Lonberg, N., et al., Nature368 (1994) 856-859; reviewed in Lonberg, N., Handbook of ExperimentalPharmacology 113 (1994) 49-101; Lonberg, N., and Huszar, D., Intern.Rev. Immunol. 25 (1995) 65-93; and Harding, F., and Lonberg, N., Ann. N.Acad. Sci 764 (1995) 536-546). The preparation of HuMab mice isdescribed in Taylor, L., et al., Nucleic Acids Research 20 (1992)6287-6295; Chen, J., et al., International Immunology 5 (1993) 647-656;Tuaillon, N., et al., Proc. Natl. Acad. Sci USA 90 (1993) 3720-3724;Choi, T. K., et al., Nature Genetics 4 (1993) 117-123; Chen, J., et al.,EMBO J. 12 (1993) 821-830; Tuaillon, N., et al., Immunol. 152 (1994)2912-2920; Lonberg, N., et al., Nature 368 (1994) 856-859; Lonberg, N.,Handbook of Experimental Pharmacology 113 (1994) 49-101; Taylor, L., etal., Int. Immunol. 6 (1994) 579-591; Lonberg, N., and Huszar, D.,Intern. Rev. Immunol. 25 (1995) 65-93; Harding, F., and Lonberg, N.,Ann. N. Acad. Sci 764 (1995) 536-546; Fishwild, D. M., et al., Nat.Biotechnol. 14 (1996) 845-851, the contents of all of which are herebyincorporated by reference in their entirety. See further, U.S. Pat. Nos.5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;5,661,016; 5,814,318; 5,874,299; 5,545,807; 5,770,429; WO 98/24884; WO94/25585; WO 93/1227; WO 92/22645; and WO 92/03918.

To generate fully human monoclonal antibodies to P-selectin, HuMab micecan be immunized with a purified or enriched preparation of P-selectinantigen and/or cells expressing P-selectin in accordance with thegeneral method, as described by Lonberg, N., et al., Nature 368 (1994)856-859; Fishwild, D. M., et al., Nat. Biotechnol. 14 (1996) 845-851 andWO 98/24884. Preferably, the mice will be 6-16 weeks of age upon thefirst immunization. For example, a purified or enriched preparation ofsoluble P-selectin antigen (e.g. purified from P-selectin-expressingcells) can be used to immunize the HuMab mice intraperitoneally. In theevent that immunizations using a purified or enriched preparation ofP-selectin antigen do not result in antibodies, mice can also beimmunized with cells expressing P-selectin, e.g., a tumor cell line, topromote immune responses. Cumulative experience with various antigenshas shown that the HuMab transgenic mice respond best when initiallyimmunized intraperitoneally (i.p.) with antigen in complete Freund'sadjuvant, followed by every other week i.p. immunizations (for example,up to a total of 6) with antigen in incomplete Freund's adjuvant. Theimmune response can be monitored over the course of the immunizationprotocol with plasma samples being obtained by retroorbital bleeds. Theplasma can be screened by ELISA, and mice with sufficient titers ofanti-P-selectin human immunoglobulin can be used for immortalization ofcorresponding B cells. Mice can be boosted intravenously with antigen 3to 4 days before sacrifice and removal of the spleen and lymph nodes. Itis expected that 2-3 fusions for each antigen may need to be performed.Several mice will be immunized for each antigen. For example, a total oftwelve HuMab mice of the HCo7 and HCo12 strains can be immunized.

The HCo7 mice have a JKD disruption in their endogenous light chain(kappa) genes (as described in Chen, J., et al., EMBO J. 12 (1993)821-830), a CMD disruption in their endogenous heavy chain genes (asdescribed in Example 1 of WO 01/14424), a KCo5 human kappa light chaintransgene (as described in Fishwild, D. M., et al., Nat. Biotechnol. 14(1996) 845-851), and a HCo7 human heavy chain transgene (as described inU.S. Pat. No. 5,770,429).

The HCo12 mice have a JKD disruption in their endogenous light chain(kappa) genes (as described in Chen, J., et al., EMBO J. 12 (1993)821-830), a CMD disruption in their endogenous heavy chain genes (asdescribed in Example 1 of WO 01/14424), a KCo5 human kappa light chaintransgene (as described in Fishwild, D. M., et al., Nat. Biotechnol. 14(1996) 845-851), and a HCo12 human heavy chain transgene (as describedin Example 2 of WO 01/14424).The mouse lymphocytes can be isolated andfused with a mouse myeloma cell line using PEG based on standardprotocols to generate hybridomas. The resulting hybridomas are thenscreened for the production of antigen-specific antibodies. For example,single cell suspensions of splenic and lymph node-derived lymphocytesfrom immunized mice are fused to one-sixth the number of SP 2/0nonsecreting mouse myeloma cells (ATCC, CRL 1581) with 50% PEG. Cellsare plated at approximately 2×10⁵ in flat bottom microtiter plate,followed by about two weeks incubation in selective medium.

Individual wells are then screened by ELISA for human anti-P-selectinmonoclonal IgM and IgG antibodies. Once extensive hybridoma growthoccurs, medium is analyzed, usually after 10-14 days. The antibodysecreting hybridomas are replated, screened again, and if still positivefor human IgG, anti-P-selectin monoclonal antibodies, can be subclonedat least twice by limiting dilution. The stable subclones are thencultured in vitro to produce antibody in tissue culture medium forcharacterization.

Because CDR sequences are responsible for antibody-antigen interactions,it is possible to express recombinant antibodies according to theinvention by constructing expression vectors that include the CDRsequences according to the invention onto framework sequences from adifferent human antibody (see, e.g., Riechmann, L., et al., Nature 332(1998) 323-327; Jones, P., et al., Nature 321 (1986) 522-525; and Queen,C., et al., Proc. Natl. Acad. See. U.S.A. 86 (1989)10029-10033). Suchframework sequences can be obtained from public DNA databases thatinclude germline human antibody gene sequences. These germline sequenceswill differ from mature antibody gene sequences because they will notinclude completely assembled variable genes, which are formed by V(D)Jjoining during B cell maturation. Germline gene sequences will alsodiffer from the sequences of a high affinity secondary repertoireantibody at individual evenly across the variable region.

The invention further comprises the use of an antibody according to theinvention for the diagnosis of P-selectin in vitro, preferably by animmunological assay determining the binding between P-selectin of asample and the antibody according to the invention.

In another aspect, the present invention provides a composition, e.g. apharmaceutical composition, containing one or a combination of humanmonoclonal antibodies, or the antigen-binding portion thereof, of thepresent invention, formulated together with a pharmaceuticallyacceptable carrier. More specifically, the composition is apharmaceutical or a diagnostic composition and even more specificallythe pharmaceutical composition comprises an antibody as defined aboveand at least one pharmaceutically acceptable excipient.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include a composition of the present inventionwith at least one agent useful in the prophylaxis or treatment a diseaseassociated with critical limb ischemia (CLI/PAOD) or other conventionaltherapy.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g. by injection or infusion).

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the antibody and does not impart anyundesired toxicological effects (see e.g. Berge, S. M., et al., J.Pharm. Sci. 66 (1977) 1-19). Such salts are included in the invention.Examples of such salts include acid addition salts and base additionsalts. Acid addition salts include those derived from nontoxic inorganicacids, such as hydrochloric salts.

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results.

To administer a compound of the invention by certain routes ofadministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the compound may be administered to a subject in anappropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions.

Pharmaceutically acceptable excipients or carriers include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is known in the art.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

These compositions may also contain excipients or adjuvants such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of presence of microorganisms may be ensured both bysterilization procedures, supra, and by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol, sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts. A typical weekly dosagemight range from about 0.1 mg/kg to about 20 mg/kg or more, depending onthe factors mentioned above.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carriercan be an isotonic buffered saline solution, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyetheylene glycol,and the like), and suitable mixtures thereof.

Proper fluidity can be maintained, for example, by use of coating suchas lecithin, by maintenance of required particle size in the case ofdispersion and by use of surfactants. In many cases, it is preferable toinclude isotonic agents, for example, sugars, polyalcohoils such asmannitol or sorbitol, and sodium chloride in the composition. Long-termabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

The invention comprises a method for the treatment of a patient in needof therapy, characterized by administering to the patient atherapeutically effective amount of an antibody which binds P-selectin,contains a Fc part derived from human origin and does not bindcomplement factor C1q.

The invention comprises the use of an antibody which binds P-selectin,contains a Fc part derived from human origin and does not bindcomplement factor C1q for therapy.

The invention comprises the use of an antibody which binds P-selectin,contains a Fc part derived from human origin and does not bindcomplement factor C1q for the preparation of a medicament for theprophylaxis and treatment of inflammatory and thrombotic disorders.

The invention comprises the use of an antibody which binds P-selectin,contains a Fc part derived from human origin and does not bindcomplement factor C1q for the treatment of PAOD and CLI.

The present invention thus provides an antibody binding to P-selectin,not binding to complement factor C1q, containing an Fc part derived fromhuman origin, and being characterized in that said antibody is anantibody of human subclass IgG1, containing at least one mutation inL234, L235, D270, N297, E318, K320, K322, P331 and/or P329 or is anantibody of human subclass IgG4 wherein S228 is replaced by P and L235is replaced by E. In one embodiment the antibody is a human antibody. Inanother embodiment the antibody is a humanized antibody.

In one embodiment the present invention provides an antibody binding toP-selectin, not binding to complement factor C1q, containing an Fc partderived from human origin, and being characterized in that said antibodyis an antibody of human subclass IgG1, containing at least one mutationin L234, L235, D270, N297, E318, K320, K322, P331 and/or P329 or is anantibody of human subclass IgG4 wherein S228 is replaced by P and L235is replaced by E, wherein non-binding of the antibody to complementfactor C1q refers to an ELISA assay measurement wherein the maximalbinding (Bmax) of C1q to the antibody at a concentration of 10 μg/ml ofthe antibody is ≦30% of Bmax of the antibody LC 1004-002 of cell linehu-Mab<P-selectin>LC 1004-002 (DSM ACC2641). In another embodiment themaximal binding is ≦20% of Bmax of the antibody LC 1004-002 of cell linehu-Mab<P-selectin>LC 1004-002 (DSM ACC2641).

In one embodiment the present invention provides an antibody binding toP-selectin, not binding to complement factor C1q, containing an Fc partderived from human origin, and being characterized in that said antibodyis an antibody of human subclass IgG1, containing at least one mutationin L234, L235, D270, N297, E318, K320, K322, P331 and/or P329 or is anantibody of human subclass IgG4 wherein S228 is replaced by P and L235is replaced by E, wherein the antibody binds to P-selectin with a K_(D)value of less than 10⁻⁸ M in a BIAcore assay. In another embodiment theK_(D) range is 10⁻¹¹ to 10⁻⁹ M.

In one embodiment the present invention provides an antibody binding toP-selectin, not binding to complement factor C1q, containing an Fc partderived from human origin, and being characterized in that said antibodyis an antibody of human subclass IgG1, containing at least one mutationin L234, L235, D270, N297, E318, K320, K322, P331 and/or P329 or is anantibody of human subclass IgG4 wherein S228 is replaced by P and L235is replaced by E, wherein the antibody binds at least 1000 fold morespecifically to P-selectin than to E- and/or L-selectin as measured byEC50 values in an ELISA assay, wherein P— and E- and/or L-selectin arecoated onto the microtiter plate. In another embodiment the EC50 valueson E- and L-selectin transfectants are above 100 μg/ml.

In one embodiment the present invention provides an antibody binding toP-selectin, not binding to complement factor C1q, containing an Fc partderived from human origin, and being characterized in that said antibodyis an antibody of human subclass IgG1, containing at least one mutationin L234, L235, D270, N297, E318, K320, K322, P331 and/or P329 or is anantibody of human subclass IgG4 wherein S228 is replaced by P and L235is replaced by E, wherein the antibody inhibits the adhesion ofleukocyte-like HL60 cells to purified P-selectin with an IC50 value ofno more than 1 μg/ml. In another embodiment the IC50 value is in therange of 0.08 to 0.5 μg/ml. In still another embodiment the IC50 valueis in the range of 0.08 to 0.11 μg/ml.

In one embodiment the present invention provides an antibody binding toP-selectin, not binding to complement factor C1q, containing an Fc partderived from human origin, and being characterized in that said antibodyis an antibody of human subclass IgG1, containing at least one mutationin L234, L235, D270, N297, E318, K320, K322, P331 and/or P329 or is anantibody of human subclass IgG4 wherein S228 is replaced by P and L235is replaced by E, wherein

-   -   (a) the adhesion of leukocyte-like HL60 cells to activated        platelets is inhibited with an IC50 value of 0.05 to 0.3 μg/ml;    -   (b) the antibody inhibits the interaction of leukocytes with a        monolayer of platelets by more than 70%;    -   (c) the antibody inhibits the adhesion of leukocytes to        activated endothelial cells in a human flow system in the range        of 60 to 90% at a concentration of 3 μg/ml;    -   (d) the antibody does not bind the C3 protein;    -   (e) the antibody does not elicit complement-dependent        cytotoxicity (CDC);    -   (f) the antibody does not bind to Fcγ receptors on NK effector        cells; or    -   (g) the antibody does not elicit antibody-dependent cellular        cytotoxicity (ADCC).

In one embodiment the present invention provides an antibody binding toP-selectin characterized in that the variable heavy chain amino acidsequence CDR3 of said antibody is selected from the group consisting ofthe heavy chain CDR3 sequences SEQ ID NO: 38, 39, 40, 41 or 42.

In one embodiment the present invention provides an antibody binding toP-selectin, comprising a variable heavy chain and a variable lightchain, characterized in that the variable heavy chain comprises CDRsequences CDR1, CDR2 and CDR3 and CDR1 being selected from the groupconsisting of SEQ ID NOs: 29, 30, 31, 32, CDR2 being selected from thegroup consisting of SEQ ID NOs: 33, 34, 35, 36, 37, CDR3 being selectedfrom the group consisting of SEQ ID NOs: 38, 39, 40, 41, 42, whereinsaid CDRs are selected independently of each other.

In one embodiment the present invention provides an antibodycharacterized in that the variable light chain comprises CDR sequencesCDR1, CDR2 and CDR3, and CDR1 is selected from SEQ ID NOs: 43, 44, CDR2is selected from SEQ ID NOs: 45, 46 and CDR3 is selected from SEQ IDNOs: 47, 48, 49, 50, 51, 52 wherein said CDRs are selected independentlyof each other.

In one embodiment the present invention provides an antibody,characterized in that said antibody binds P-selectin and that theantibody comprises a variable region independently selected from thegroup consisting of

-   -   a) the light chain variable domain defined by amino acid        sequence SEQ ID NO:1 and the heavy chain variable domain defined        by SEQ ID NO:2;    -   b) the light chain variable domain defined by amino acid        sequence SEQ ID NO:3 and the heavy chain variable domain defined        by SEQ ID NO:4;    -   c) the light chain variable domain defined by amino acid        sequence SEQ ID NO:5 and the heavy chain variable domain defined        by SEQ ID NO:6;    -   d) the light chain variable domain defined by amino acid        sequence SEQ ID NO:7 and the heavy chain variable domain defined        by SEQ ID NO:8;    -   e) the light chain variable domain defined by amino acid        sequence SEQ ID NO:9 and the heavy chain variable domain defined        by SEQ ID NO:10;    -   f) the light chain variable domain defined by amino acid        sequence SEQ ID NO:11 and the heavy chain variable domain        defined by SEQ ID NO:12;    -   g) the light chain variable domain defined by amino acid        sequence SEQ ID NO:13 and the heavy chain variable domain        defined by SEQ ID NO:14;    -   h) the light chain variable domain defined by amino acid        sequence SEQ ID NO:15 and the heavy chain variable domain        defined by SEQ ID NO:16;    -   i) the light chain variable domain defined by amino acid        sequence SEQ ID NO:17 and the heavy chain variable domain        defined by SEQ ID NO:18;    -   j) the light chain variable domain defined by amino acid        sequence SEQ ID NO:19 and the heavy chain variable domain        defined by SEQ ID NO:20; and    -   k) the light chain variable domain defined by amino acid        sequence SEQ ID NO:21 and the heavy chain variable domain        defined by SEQ ID NO:22.

In one embodiment the present invention provides an antibody binding toP-selectin, not binding to complement factor C1q, containing an Fc partderived from human origin, and being characterized in that said antibodyis an antibody of human subclass IgG1, containing at least one mutationin L234, L235, D270, N297, E318, K320, K322, P331 and/or P329 or is anantibody of human subclass IgG4 wherein S228 is replaced by P and L235is replaced by E, wherein the antibody comprises the CDR1, CDR2 and CDR3regions of the light chain variable domain defined by amino acidsequence SEQ ID NO:3 and the CDR1, CDR2 and CDR3 regions of the heavychain variable domain defined by SEQ ID NO:4. In another embodiment theantibody comprises the light chain variable domain defined by amino acidsequence SEQ ID NO:3 and the heavy chain variable domain defined by SEQID NO:4.

In one embodiment the present invention provides an antibody binding toP-selectin, not binding to complement factor C1q, containing an Fc partderived from human origin, and being characterized in that said antibodyis an antibody of human subclass IgG1, containing at least one mutationin L234, L235, D270, N297, E318, K320, K322, P331 and/or P329 or is anantibody of human subclass IgG4 wherein S228 is replaced by P and L235is replaced by E, wherein

-   -   (a) the antibody comprises at least one amino acid mutation in        the Fc part causing non-binding to complement factor C1q;    -   (b) the human heavy chain constant region comprises the amino        acid sequence independently selected from the group consisting        of SEQ ID NO: 25, SEQ ID NO:26 and 28;    -   (c) the antibody comprises a K-light chain constant region as        defined by SEQ ID NO:23;    -   (d) the antibody comprises at least one amino acid mutation        causing non-binding to complement C1q;    -   (e) the antibody comprises a heavy chain constant region        selected from the group consisting of IgG1v1, IgG1v2 and IgG4v1;        or    -   (f) the antibody is a Fab, F(ab′)₂ or a single-chain fragment.

In one embodiment the present invention provides an anti-P selectinantibody, characterized in that it a) is a human or humanized antibody,and b) binds at least 1000 fold more specifically to P-selectin than toE- or L-selectin as measured by EC50 values in an ELISA assay, whereinP— and E- and/or L-selectin are coated onto the microtiter plate. Inanother embodiment the antibody comprises the amino acid sequence asdefined by SEQ ID NO:24, 25 or 26 γ1 heavy chain constant region or SEQID NO:27 or 28 γ4 heavy chain constant region. In still anotherembodiment the antibody is produced by a cell line selected from thegroup consisting of hu-Mab<P-selectin>LC 1004-001 (DSM ACC2640),hu-Mab<P-selectin>LC 1004-002 (DSM ACC2641) and hu-+Mab<P-selectin>LC1004-017(DSM ACC2642).

It is to be understood that the invention provides the embodiments withthe definitions as described in paragraphs above-.

The following examples, references, sequence listing and figures areprovided to aid the understanding of the present invention, the truescope of which is set forth in the appended claims. It is understoodthat modifications can be made in the procedures set forth withoutdeparting from the spirit of the invention.

DESCRIPTION OF THE SEQUENCE LISTING

-   SEQ ID NO:1 LC1004-001 light chain, variable domain of HuMab    1004-001-   SEQ ID NO:2 LC1004-001 heavy chain, variable domain of HuMab    1004-001-   SEQ ID NO:3 LC 1004-002 light chain, variable domain of HuMab 002-   SEQ ID NO:4 LC 1004-002 heavy chain, variable domain of HuMab 002-   SEQ ID NO:5 LC 1004-003 light chain, variable domain of HuMab 003-   SEQ ID NO:6 LC 1004-003 heavy chain, variable domain of HuMab 003-   SEQ ID NO:7 LC 1004-004 light chain (I), variable domain of HuMab    004 (I)-   SEQ ID NO:8 LC 1004-004 heavy chain (I), variable domain of HuMab    004 (I)-   SEQ ID NO:9 LC 1004-004 light chain (II), variable domain of HuMab    004 (II)-   SEQ ID NO:10 LC 1004-004 heavy chain (II), variable domain of HuMab    004 (II)-   SEQ ID NO:11 Light chain, variable domain of HuMab 005-   SEQ ID NO:12 Heavy chain, variable domain of HuMab 005-   SEQ ID NO:13 Light chain, variable domain of HuMab 010 (I)-   SEQ ID NO:14 Heavy chain, variable domain of HuMab 010 (I)-   SEQ ID NO:15 Light chain, variable domain of HuMab 010 (II)-   SEQ ID NO:16 Heavy chain, variable domain of HuMab 010 (II)-   SEQ ID NO:17 Light chain, variable domain of HuMab 010 (III)-   SEQ ID NO:18 Heavy chain, variable domain of HuMab 010 (III)-   SEQ ID NO:19 Light chain,variable domain of HuMab 011-   SEQ ID NO:20 Heavy chain, variable domain of HuMab 011-   SEQ ID NO:21 Light chain, variable domain of HuMab 017-   SEQ ID NO:22 Heavy chain, variable domain of HuMab 017-   SEQ ID NO:23 κ light chain constant region-   SEQ ID NO:24 γ1 heavy chain constant region-   SEQ ID NO:25 γ1 heavy chain constant region PVA236/GLPSS331 (IgG1v1)-   SEQ ID NO:26 γ1 heavy chain constant region L234A/L235A (IgG1v2)-   SEQ ID NO:27 γ4 heavy chain constant region-   SEQ ID NO:28 γ4 heavy chain constant region S228/L235E (IgG4v1)-   SEQ ID NO:29-32 Heavy chain CDR1-   SEQ ID NO:33-37 Heavy chain CDR2-   SEQ ID NO:38-42 Heavy chain CDR3-   SEQ ID NO:43-44 Light chain CDR1-   SEQ ID NO:45-46 Light chain CDR2-   SEQ ID NO:47-52 Light chain CDR3

ABBREVIATIONS

Amino acids are abbreviated either in the three (Leu) or one letter code(L) Antibody HuMab 00X is also named antibody 00X

-   L234 means amino acid leucine at position 234 according to EU    numbering (Kabat)-   L234A means amino acid leucine at position 234 is changed to alanine-   PVA236 means that in the 236 region ELLG of IgG1 or EFLG of IgG4 is    amended in PVA-   GLPSS331 means that in the 331 region ALPAP of IgG1 or GLPAP of IgG2    is changed to GLPSS-   Amendments in the other IgG subclasses analogously

EXAMPLES Generation of a Hybridoma Cell Line Producing Anti-P-SelectinAntibodies

Culture of Hybridomas

HuMab hybridomas were cultured in IMDM (Cambrex), Fetal clone 1 Bovineserum (Perbio Science), origin Hybridoma cloning factor (Igen), sodiumpyruvate, penicillin/streptomycin, 2-mercaptoethanol, HAT(Sigma-Aldrich) and Kanamycin (Invitrogen) in 37° C. and 5% CO₂.

Generation of a Hybridoma Cell Line Producing Anti-P-Selectin AntibodiesImmunization Procedure of Transgenic Mice

Protocol A:

10 HCo7 transgenic mice (5 males and 5 females), strain GG2201 (Medarex,San Jose, Calif., USA) were immunized with a recombinant truncated formof P-selectin which lacks the transmembrane and cytoplasmic domain ofP-selectin and which was purchased from R&D Systems. For the firstimmunization 50 μg recombinant P-selectin, dissolved in 100 μl PBS, wasmixed with 100 μl complete Freunds' adjuvant. For the remainingimmunizations recombinant P-selectin coupled to KLH was used. For thesecond immunization 50 μg KLH-coupled recombinant P-selectin wasdissolved in 100 μl PBS and mixed with 100 μl incomplete Freunds'adjuvant. For the remaining immunizations 20 μg KLH-coupled recombinantP-selectin was dissolved in 100 μl PBS and mixed with 100 μl incompleteFreunds' adjuvant. Immunizations were administered alternatinginterperitoneal and subcutaneous starting with an interperitonealimmunization.

Protocol B:

3 HCo7 (all female) and 3 KM (all male) transgenic mice, strain GG2489(Medarex, San Jose, Calif., USA) were immunized with full-lengthP-selectin purified from human outdated platelets by immunoaffinitychromatography (s. below). For the first immunization, 50 μg of thepurified P-selectin, dissolved in 100 μl PBS, was mixed with 100 μlcomplete Freunds' adjuvant (CFA; Difco Laboratories, Detroit, USA). Forthe second immunization, 50 μg of the purified P-selectin, dissolved in100 μl PBS, was mixed with 100 μl incomplete Freunds' adjuvant (ICFA;Difco).

For all other immunizations, 20 μg of the purified P-selectin was usedand mixed with 100 μl incomplete Freunds' adjuvant.

Antigen Specific ELISA

Anti-P-selectin titers in sera of immunized mice were determined byantigen specific ELISA. Plate (96 flat bottom ELISA plate, Greiner) wascoated with 0.1 μg/ml purified P-selectin dissolved in PBS and coatedovernight at room temperature. Thereafter, wells were blocked with PBSTC(PBS containing 0.05% Tween 20 (Sigma-Aldrich Chemie BV) and 2%chickenserum (Gibco)) for 1 hour at room temperature.

Tested serum taps were diluted 1:100 in PBSTC and added to the wells.Serum obtained from mice prior to immunization was dissolved 1:100 inPBSTC and used as negative control. A mouse antibody directed againsthuman P-selectin ( 1/7, produced in house by Roche Basel) was dissolved1:100 in PBSTC and used as a positive control. Plates were incubated for1 hour at room temperature. Subsequently, plates were washed twice usingPBST (PBS containing 0.05% Tween 20. Gt-α-huIgG-HRP (Jackson) wasdiluted 1:5000 in PBSTC and added to the wells containing the testedtaps and the negative control. Rb-α-mIgG (Jackson) was diluted 1:3000 inPBSTC and added to the wells containing the positive control. Plateswere incubated for 1 hour at room temperature. Finally, plates werewashed twice using PBSTand developed with freshly prepared ABTS®solution (1 mg/ml) (ABTS: 2,2′-azino bis(3-ethylbenzthiazoline-6-sulfonic acid) for 30 minutes at roomtemperature (RT) in the dark. Absorbance was measured at 405 nm.

Boosting of Mice

When serum titers of anti-P-selectin were sufficient, mice wereadditionally boosted twice with 20 μg recombinant human P-selectin in100 μl PBS, intraveneously 4 and 3 days before fusion.

Hybridoma Generation

Mice were sacrificed and the spleen and lymph nodes flanking theabdominal aorta and vena cava were collected. Fusion of splenocytes andlymph node cells with the fusion partner SP 2.0 cells was performedaccording to standard operating procedures.

Human monoclonal antibodies with variable heavy and light sequences ofSEQ ID NOs 1-22 were obtained by the immunization procedure.

κ-ELISA

To determine whether hybridomas that resulted from the fusion generatehuman antibodies, a κ-ELISA was performed. ELISA plates were coated withrat anti-human IgG κ-light chain antibody (DAKO) diluted 1/10000 in PBSby overnight incubation at 4° C. After discarding the wells, plates wereblocked by incubation with PBSTC for 1 hour at room temperature.Thereafter, wells were incubated with hybridoma culture supernatant, ½diluted in PBSTC. Culture medium ½ diluted in PBSTC was used as negativecontrol, κ-light positive mouse serum 1/100 diluted in PBSTC served aspositive control. Subsequently, wells were washed thrice and wereincubated with HRP-conjugated rat anti-human IgG F(ab′)₂ (DAKO), diluted1/2000 in PBSTC for 1 h at 37° C. Wells were washed thrice and assayswere developed with freshly prepared ABTS® solution (1 mg/ml) for 30minutes at room temperature (RT) in the dark. Absorbance was measured at405 nm in an ELISA plate reader.

Cloning and Sequence Analysis of Anti-P-Selectin HuMab Variable Domains(κ-Light and γ1-Heavy Chains)

The nucleotide sequences coding for the light chain variable regionV_(L) and the heavy chain variable region V_(H) of the P-selectin HuMabswere isolated by a standard cDNA synthesis/PCR procedure.

Total RNA was prepared from 1×10⁶-1×10⁷ hybridoma cells using theRNeasy® Mini Kit (Qiagen). Hybridoma derived RNA was used as a templatefor the 1^(st) strand cDNA synthesis which was performed according to aconventional method making use of an oligo dT primer. 2^(nd)-strand cDNAsynthesis and further PCR amplification of V_(L) and V_(H) encoding cDNAfragments were performed with revers light and heavy chain primerscomplementary to nucleotide sequences of the κ-light and γ1-heavy chainconstant region and 5′-specific light and heavy chain primers,respectively. The PCR products were cloned using the TOPO TA cloning kitfrom Invitrogen™ life technologies and pCR4-TOPO as a cloning vector.Cloned PCR products were identified by restriction mapping of theappropriate plasmids using EcoRI for digestion and expected/calculatedDNA fragment sizes of about 740 and 790 bp for V_(L) and V_(H),respectively.

The DNA sequence of cloned PCR fragments was determined by double strandsequencing.

The GCG (Genetics Computer Group, Madison, Wis.) software packageversion 10.2 was used for general data processing. DNA and proteinsequences were aligned using the GCG modul CLUSTALW. Sequence alignmentswere tabulated, edited and color-coded using the program GENEDOC(version 2.1).

Construction of Expression Plasmids for an Anti-P-Selectin IgG1 HuMab

The anti-P-selectin HuMab light and heavy chain encoding genes wereseparately assembled in mammalian cell expression vectors.

Thereby the gene segments encoding the anti-P-selectin HuMab light chainvariable region (V_(L)) and the human κ-light chain constant region(C_(L)) were joined as were gene segments for the anti-P-selectin HuMabheavy chain variable region (V_(H)) and the human γ1-heavy chainconstant region (C_(H1)-Hinge-C_(H2)-C_(H3)).

General information regarding the nucleotide sequences of human lightand heavy chains from which the codon usage can be deduced is given in:Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S., and Foeller, C.(1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIHPublication No 91-3242.

The transcription unit of the anti-P-selectin HuMab κ-light chain iscomposed of the following elements:

-   -   The immediate early enhancer and promoter from the human        cytomegalovirus (HCMV),    -   A synthetic 5′-UT including a Kozak sequence,    -   A murine immunoglobulin heavy chain signal sequence including        the signal sequence intron,    -   The cloned anti-P-selectin HuMab variable light chain cDNA        arranged with a unique BsmI restriction site at the 5′ end and a        splice donor site and a unique NotI restriction site at the 3′        end,    -   The genomic human κ-gene constant region, including the intron 2        mouse Ig-κ enhancer [Picard, D., and Schaffner, W. (1984) Nature        307, 80-82] and    -   The human immunoglobulin κ-polyadenylation (“poly A”) signal        sequence.

The transcription unit of the anti-P-selectin HuMab γ1-heavy chain iscomposed of the following elements:

-   -   The immediate early enhancer and promoter from the human        cytomegalovirus (HCMV),    -   A synthetic 5′-UT including a Kozak sequence,    -   A modified murine immunoglobulin heavy chain signal sequence        including the signal sequence intron,    -   The cloned anti-P-selectin HuMab variable heavy chain cDNA        arranged with a unique BsmI restriction site at the 5′ and a        splice donor site and a unique NotI restriction site at the 3′        end,    -   The genomic human γ1-heavy gene constant region, including the        mouse Ig μ-enhancer [Neuberger, M. S. (1983) Embo J 2,        1373-1378],    -   The human γ1-immunoglobulin polyadenylation (“poly A”) signal        sequence.

Functional elements of the anti-P-selectin HuMab κ-light chain andγ1-heavy chain expression plasmids: Beside the anti-P-selectin HuMabκ-light chain or γ1-heavy chain expression cassette these plasmidscontain

-   -   A hygromycin resistance gene    -   An origin of replication, oriP, of Epstein-Barr virus (EBV)    -   An origin of replication from the vector pUC18 which allows        replication of this plasmid in E. coli, and    -   A β-lactamase gene which confers ampicillin resistance in E.        coli.

Construction of Expression Plasmids for an Anti-P-Selectin IgG4 HuMab

An anti-P-selectin γ4-heavy chain prototype expression plasmid wasderived from the anti-P-selectin γ1-heavy chain expression plasmid byreplacing the human genomic γ1-constant region and γ1-immunoglobulinpolyadenylation (“poly A”) signal sequence by the human genomicγ4-constant region and γ4-immunoglobulin polyadenylation-signalsequence.

For the expression of anti-P-selectin HuMab κ-light chains the sameexpression plasmids were used as described for IgG1 (see above).

Construction of Expression Plasmids for Mutant (Variant) Anti-P-SelectinIgG1 and IgG4

Expression plasmids encoding mutant anti-P-selectin γ1- and γ4-heavychains were created by site-directed mutagenesis of the wild typeexpression plasmids using the QuickChange™ Site-Directed mutagenesis Kit(Stratagene).

The following mutants were generated for LC1004-002. Amino acids arenumbered according to EU numbering [Edelman, G. M., Cunningham, B. A.,Gall, W. E., Gottlieb, P. D., Rutishauser, U., and Waxdal, M. J. (1969)Proc Natl Acad Sci U S A 63, 78-85; Kabat, E. A., Wu, T. T., Perry, H.M., Gottesman, K. S., and Foeller, C. (1991) Sequences of Proteins ofImmunological Interest, Fifth Ed., NIH Publication No 91-3242]. TABLE 1Isotype Abbreviation Mutations Description IgG1 IgG1v1 PVA-236; Theamino acid sequence GLPSS331 Glu₂₃₃Leu₂₃₄Leu₂₃₅Gly₂₃₆ of the human γ1-as specified heavy chain is replaced by the amino acid by sequencePro₂₃₃Val₂₃₄Ala₂₃₅ of the human E233P; γ2-heavy chain. L234V; The aminoacid sequence L235A; delta Ala₃₂₇Leu₃₂₈Pro₃₂₉Ala₃₃₀Pro₃₃₁ of the G236;human γ1-heavy chain is replaced by the A327G; amino acid sequenceA330S; Gly₃₂₇Leu₃₂₈Pro₃₂₉Ser₃₃₀Ser₃₃₁ of the human P331S γ4-heavy chainSEQ IDNO: 25 IgG1 IgG1v2 L234A; The amino acid sequence Leu₂₃₄Leu₂₃₅ ofL235A the human γ1-heavy chain is replaced by SEQ the amino acidsequence Ala₂₃₄Ala₂₃₅ IDNO: 26 IgG4 IgG4v1 S228P; Ser₂₂₈ of the humanγ4-heavy chain is L235E replaced by Pro₂₂₈ and Leu₂₃₅ of the SEQ humanγ4-heavy chain is replaced by IDNO: 28 Glu₂₃₅

Production of Recombinant Anti P-Selectin HuMabs

Recombinant HuMabs were generated by transient transfection of adherentHEK293-EBNA cells (ATTC # CRL-10852) cultivated in DMEM (Gibco)supplemented with 10% ultra-low IgG FCS (Gibco), 2 mM Glutamine (Gibco),1% v/v nonessential aminoacids (Gibco) and 250 μg/ml G418 (Roche). Fortransfection Fugene™ 6 (Roche) Transfection Reagent was used in a ratioof reagent (μl) to DNA (μg) ranging from 3:1 to 6:1. Immunoglobulinlight and heavy chains were expressed from two different plasmids usinga molar ratio of light chain to heavy chain encoding plasmid from 1:2 to2:1. HuMab containing cell culture supernatants were harvested at day 4to 11 after transfection. Supernatants were stored at −20° C. untilpurification.

General information regarding the recombinant expression of humanantibody in e.g. HEK293 is given in: Meissner, P., Pick, H., Kulangara,A., Chatellard, P., Friedrich, K., and Wurm, F. M. (2001) BiotechnolBioeng 75, 197-203.

Determination of the Affinity of Anti-P-Selectin HuMabs

Equipment:

-   Instrument: BIACORE® 2000-   Chip: CM5-   Coupling: amine coupling-   Buffer: HBS (HEPES, NaCl), pH 7.4, 25° C.

For affinity measurements rabbit anti human Fcγ antibodies (Dianova)have been coupled by amine coupling to the chip surface for presentationof the antibody against P-selectin. Approximately 400 RU of antiP-selectin antibodies were bound. Recombinant P-selectin (R&D Systems)was added in various concentrations between 0-50 nM. Association wasmeasured by P-selectin-injection for 120 seconds; dissociation wasmeasured by washing the chip surface with buffer for 180 seconds. Theaffinity data for different P-selectin antibodies are shown in Table 2.Using Biaevaluation Software the kinetic data were fitted to a 1:1Langmuir binding model of P-selectin to the presented monoclonalantibody. TABLE 2 Affinity data measured by SPR (BIACORE ® 2000)Antibody HuMab ka (1/Ms) kd (1/s) KA (1/M) KD (M) 001 6.08 × 10⁵ 4.19 ×10⁻⁴ 1.45 × 10⁹ 6.89 × 10⁻¹⁰ 002 8.10 × 10⁵ 2.13 × 10⁻³ 3.81 × 10⁹ 2.63× 10⁻⁹  003 6.60 × 10⁵ 2.91 × 10⁻⁴ 2.27 × 10⁹ 4.41 × 10⁻¹⁰ 005 8.42 ×10⁵ 2.89 × 10⁻⁴ 2.91 × 10⁹ 3.43 × 10⁻¹⁰ 011 1.77 × 10⁶ 2.38 × 10⁻³ 7.44× 10⁸ 1.34 × 10⁻⁹  012 1.08 × 10⁶ 1.25 × 10⁻⁴ 8.65 × 10⁹ 1.16 × 10⁻¹⁰013 1.46 × 10⁶ 2.02 × 10⁻⁴ 7.22 × 10⁹ 1.39 × 10⁻¹⁰ 017 7.79 × 10⁵ 1.39 ×10⁻⁵ 5.59 × 10⁹ 1.79 × 10⁻¹¹

Inhibitory Activity of the P-Selectin Antibodies in a Cell-BasedAdhesion and Rosetting Assay

Materials and Methods:

Cell adhesion assay: In the adhesion assay the effect of the HuMabs onthe adhesion of leukocyte-like HL60 cells (ATCC CCL 240) to P-selectincoated onto microtiter plates was evaluated. The HL60 cells werelabelled with BCECF-AM (2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethylester; Cat.no 216254, Calbiochem). Full-length purified P-selectin(purification procedure s. above) at a concentration of 1 μg/ml inbuffer containing 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 20 mM Tris (pH7.4) plus 0.0005% Tx100 was coated overnight at 4° C. to 96 wells plates(Nunc Immunoplate Maxisorp F96). Thereafter, the wells were blocked withthe above-mentioned buffer containing 3.5% bovine serum albumin (BSA,Fluka) for 2 h at room temperature (RT). The wells were preincubatedwith 50 μl of different dilutions of the P-selectin HuMabs or referencemouse P-selectin antibodies (WAPS 12.2, respective hybridoma cell lineprovided by ATCC) in the above-mentioned buffer containing 1% BSA for 20minutes at RT. The labelled HL60 cells (50 μl, 70,000 cells/well) wereadded and allowed to bind for 45 min at RT. In some experiments the HL60cells were preincubated with 20 μg/ml of human IgG1 for 30 minutes priorto their addition to the wells in order to block Fc receptors. Afterremoval of the unbound HL60 cells by gentle washing (4 times with theabove-mentioned buffer), the adherent cells were lysed with 120 μl NP-40(Fluka; 1% in H₂O). 100 μl of the supernates were transferred to platesto measure the respective fluorescence at an excitation wavelength of485 nm and an emission of 538 nm using a luminescence spectrometer LS50B (Perkin Elmer).

Rosetting assay: To evaluate the effect of the antibodies on theinteraction of activated platelets with HL60 cells a rosetting assay(Jungi et al., Blood 67:629 (1986)) in combination with double colorcytofluorimetric analysis (Evangelista et al., Blood 88:4183 (1996) wasapplied. Washed human platelets were prepared as described (Fox et al,Methods Enzymol 215:45 (1992)).They were activated with thrombin (finalconc 1 U/ml) for 5 min and labelled with a FITC-conjugated anti-humanGPIIb antibody pl-36 (Kouns et al., J Biol Chem 267:18844 (1992)). Then1.4-2×10⁶ platelets within 70 μl of tyrode solution were incubated withdifferent dilutions of HuMabs (100 μl) in the dark for 30 min at RT. 50μl of HL60 cell suspension (in tyrode solution) adjusted to 20×10⁶/mlwas added. The HL60 cells were labeled by incubation with 20 μl of a PE(phycoerythrin)-conjugated anti-human CD45 Ab (Code No. 555483,Pharmingen). After having incubated the labeled HL60 cells with theplatelets and the HuMabs for 30 min at room temperature in FACS tubes(Becton Dickinson), the formation of mixed aggregates or rosettes wasanalyzed by measuring both platelet and HL60 cell marker fluorescenceusing a FACScan (Becton Dickinson). Forward and side scatter, as well asgreen (FITC) and red (PE) signals were acquired by logarithmicamplification with excitation wavelength of 488 nm and emissionwavelength of 530 nm (FITC) and 570 nm (PE), respectively. Electroniccompensation was used to remove spectral overlap. HL60 cells wereidentified on the basis of forward and side scatter. Gating on eventsidentified as HL60 cells was performed to exclude single platelets. Fivethousand HL60 cell-gated events were measured for each sample. A samplein which non-activated or thrombin-activated platelets were mixed withHL60 cells in the presence of EDTA (10 mmol/l) was used to set athreshold on the green fluorescence scale. The percentage of HL60 cellsabove the threshold represents the percentage of HL60 cell bindingplatelets. The shift of the platelet marker fluorescence towards lowerfluorescence values reflects the reduction of the number of mixedaggregates with a higher number of adhering platelets in favor of anincrease of the number of mixed aggregates with a low number of adheringplatelets.

Results:

In the HL60 cell adhesion assay the P-selectin antibodies inhibited theadhesion of the HL60 cells to purified P-selectin with IC50 values inthe range of 0.08-0.5 μg/ml, Although the mutations were introduced inthe Fc portion of the antibody, both the IgG4 and IgG1 variants ofHuMabs were more potent than the parent antibody with IC50 values of0.08-0.11 μg/ml as illustrated in FIG. 1. When preincubating the HL60cells with human IgG1, the potency of the parent non-mutated antibodiesis also increased with an about 3 to 4-fold reduction of the IC50 value,as demonstrated for HuMab 002 in FIG. 1. This finding suggests that theincreased efficacy of the mutants in the adhesion assay is primarily dueto the elimination of the adhesion of the HL60 cells to P-selectin viathe Fc portion of the antibody to the Fcγ receptors.

In the rosetting assay evaluating the adhesion of human activatedplatelets expressing P-selectin to HL60 cells the IC50 values of theHuMabs were even below those of the adhesion assay due to the lowernumber of P-selectin receptors in this assay (IC50: 0.05-0.3 μg/ml,preferably between 0.05 and 0.2 μg/ml). The efficacy of the Fc variantsof the respective HuMabs tends to be increased as compared to thenon-mutated parent antibody (FIG. 2). Preincubation of the HL60 cellswith IgG1 and IgG4 prior to the incubation with the activated plateletsdid not significantly affect the inhibitory activity of both the mutantsand the parent antibody, indicating a less pronounced role of the Fcγreceptor-mediated binding in the resetting assay as compared to theadhesion assay.

Cross-Reactivity of the P-Selectin Antibodies with P-Selectin fromAnimal Species

Materials and Methods: The cross-reactivity of the P-selectin HuMabs wasevaluated by measuring (i) the binding of the HuMabs to activatedplatelets from rat and cynomologus monkey using FACS analysis and (ii)their inhibitory activity in the resetting assay evaluating the adhesionof rat and cynomologus platelets to HL60 cells.

To measure the binding of the HuMabs to activated rat and cynomologusplatelets, washed rat and cynomologus platelets were prepared similar topreparing washed human platelets (s. above). They were activated withthrombin (final conc 1 U/ml) for 5 min. Activated platelets wereincubated with different dilutions of the HuMabs (20 μl) for 30 min atRT. After binding of the HuMabs the platelets were fixed with PFA 2% atRT for 15 min. Samples were washed with Tyrode buffer and resuspended in300 ml Tyrode. The binding of the HuMabs was detected with aFITC-conjugated F(ab′)₂ fragment of rabbit anti-human IgG (Code No.F0056, Dako). As a control antibody inhibiting rat P-selectin a rabbitanti-human polyclonal anti-P-selectin antibody (Code No. 09361A,Pharmingen) was used.

To measure the inhibitory effect of the P-selectin HuMabs in theresetting assay, washed rat and cynomologus platelets were prepared asdescribed above for human platelets. The rosetting assay was performedessentially as described for human platelets. For the labelling of thecynomologus platelets the FITC-conjugated anti-human GPIIb antibodypl-36 was used, whereas the rat platelets were labeled with theFITC-conjugated mouse anti-rat CD61 antibody (Code No. 554952,Pharmingen).

Results: None of the P-selectin specific antibodies of the inventionwhich inhibit human P-selectin-mediated functions was shown to bind torat P-selectin or to inhibit the formation of mixed aggregatesconsisting of rat platelets and HL60 cells, as shown for some examplesin FIG. 3 a. However, the P-selectin HuMabs bind to and inhibitcynomologus P-selectin (FIG. 3 b).

Selectivity of the P-Selectin Antibodies vs E- and L-Selectin

Materials and Methods: The selectivity of the P-selectin HuMabs vs E-and L-selectin was determined in a cell-free ELISA measuring the bindingof the antibodies to recombinant E- and L-selectin (ADP1 and ADP2, R&DSystems) and a cell-based ELISA measuring the binding of the antibodiesto E-selectin-CHO transfectants and L-selectin-300.19 transfectants(transfectants were generated as described in Goetz et al., J Cell Biol137:509 (1997); Ley et al., Blood 82:1632 (1993)).

In the cell-free ELISA recombinant P—, E-, or L-selectin at aconcentration of 1 μg/ml in buffer containing 150 mM NaCl, 1 mM CaCl₂, 1mM MgCl₂, 20 mM Tris (pH 7.4) plus 0.0005% Tx100 was coated overnight at4° C. to 96 well plates (Nunc Immunoplate Maxisorp F96). Thereafter, thewells were blocked with the above-mentioned buffer containing 3.5%bovine serum albumin (BSA, Fluka) for 2 h at RT. The wells werepreincubated with 50 μl of different dilutions of the P-selectin HuMabsor reference mouse P—, E-selectin antibody (BBA26; R&D Systems) and goatL-selectin antibody (AF728; R&D Systems) in the above-mentioned buffercontaining 1% BSA overnight at RT. The binding of the HuMabs wasdetected by using a biotinylated anti-human IgG (Amersham, RPN1003,Final concentration 1:1000) or for the control antibodies thecorresponding biotinylated anti-mouse or anti-goat IgG. After 1 hincubation, the wells were washed (3 times) with the above-mentionedbuffer, and 0.1 ml of streptavidin-biotinylated peroxidase complex(Amersham, RPN1051), diluted 1:750 in the mentioned buffer containing0.1% BSA was added for 30 min. The wells were then washed and 0.2 ml ofperoxidase substrate solution containing ABTS(2.2′-azino-di-(3-ethylbenzthiazoline sulfonate, Boehringer, Mannheim)was added (ABTS stock solution: 1 ml 40 mM ABTS, 5 μl 30% H₂O₂ and 20 ml0.1M Na-Acetat, 0.05 NaH2PO4). The reaction was stopped after around 10min using 50 μl of 0.1 M citrate and 0.01% NaN3. The color reaction wasread at 405 nm.

In the cell-based ELISA P— and E-selectin-CHO-transfectants, afterdetaching the cells with cell-dissociation solution (Sigma C5914), wereseeded into each well of 96 well plates (TC Microwell F96 Nunc 167008)adjusted to 100,000 cells/well and cultivated in respective mediaovernight at 37° C. (medium for P— CHO-transfectants: DMEM+10% FCS+2 mMGlutamine+Penicillin 100 U/ml/Streptomycin 100 μg/ml; medium forE-selctin transfecttants: HAM F-12+10% FCS+2 mM Glutamine+Penicillin 100U/ml/Streptomycin 100 μg/ml+0.1% Fungizone+100 μg/ml Neomycin). Afterremoval of the media and blocking the wells with A-T buffer (150 mMNaCl, 1 mM CaCl₂, 1 mM MgCl₂, 20 mM Tris (pH 7.4)) containing 3%TopBlock (Code No. TB232010; Juro) for 1 h, 50 μl of different dilutionsof the P-selectin HuMabs or reference mouse P— and E-selectin antibody(s. above) in the above-mentioned buffer containing 1% TopBlock and 0.1%azide were added and incubated for 60 min at RT. After washing the wells(4 times), the bound antibodies were detected using the same steps asmentioned above for the cell-free ELISA.

Since the L-selectin 300.19 cells are suspension cells, the cell-basedELISA format had to be modified by plating the L-selectin-300.19transfectants into wells of 96 well polystyrene filter plates (Corning3510). Using the filter plates blocking and incubation solutions wereremoved by filtering them through the bottom of the plates, butotherwise the protocol was similar to that using P— and E-selectin-CHOcells. As controls non-transfected CHO and 300.19 were used.

Results:

The antibodies of the invention were highly selective vs E- andL-selectin. They bound to P-selectin-CHO cells with EC50 values in therange of 0.01 to 0.08 μg/ml, preferably in the range of 0.01 to 0.04μg/ml, whereas the EC50 values on E-selectin-CHO cells andL-selectin-300.19 were dearly above 50 μg/ml, preferably above 100μg/ml. HuMab 002 had highest selectivity with a selectivity factor vs E-and L-selectin of more than 4,000 fold in the cell-based ELISA.Furthermore HuMab 002 does not bind to E- and L-selectin transfectantsabove baseline levels up to a concentration of 100 μg/ml. Theselectivity of the Fc variants IgG4v1 and IgG1v1 of HuMab 002 is similarto that of the parent HuMab 002 (FIG. 4 a-c).

Ex Vivo Inhibitory Activity of P-Selectin Antibodies in a Fully HumanBlood Flow System

Effect of P-Selectin HuMabs on Leukocyte Adhesion to a PlateletMonolayer

Materials and Methods:

To address the effect of the P-selectin antibodies on the recruitment ofleukocytes to sites of vessel wall injury and platelet thrombi, a humanblood flow system which allows the measurement of the interaction ofhuman leukocytes with human platelets at different shear rates was usedessentially as described (Kirchhofer et al., Blood 89:1270 (1997)). In aparallel plate perfusion device human whole blood drawn from theantecubital vein of a healthy donor was perfused over a collagen surfacesimulating an injured denuded vessel wall. Collagen-coated coverslipswere prepared as described (Kirchhofer et al., Blood 89:1270 (1997)).They were positioned in three parallel plate perfusion chambers. Toallow the measurement of different shear rates (65/s and 280/s)different dimensions of perfusion chambers were used and the blood wasperfused over the collagen-coated coverslips at a constant blood flow of1 ml/min which was controlled by individual roller pumps positioneddistal to the perfusion device. Immediately after drawing the blood fromthe vein and separating the blood into three tubings, a GPIIb/IIIainhibitor (0.5 μmol/lamifiban) is added to prevent platelet aggregationand to generate platelet monolayers. At the same time, the P-selectinantibodies (the HuMabs, mutants, respective reference antibodies orhuman IgG1 and IgG4 as controls) were administered at differentconcentrations and the blood-inhibitor mixture then entered theperfusion chamber containing the collagen-coated coverslips. After a 5.5minute perfusion period, PBS is perfused through the perfusion chamberwithout interrupting the flow for 3 min. After a brief interruption offlow the chambers were fixed with 3% paraformaldehyde in PBS at 1 ml/minfor 2 min. Then the coverslips were removed from the chambers, fixedagain for 1 h in 3% paraformaldehyde in PBS at 4° C. and stored inPBS-0.03% sodium azide. To evaluate the number of leukocytes adhering tothe platelet monolayer, after air-drying the coverslips were stainedwith Diff-Quick solution (Dade Behring AG) and embedded in Merckoglas(Merck, Germany). An image analysis system (MCID, Imaging Research Inc.)was used to determine the number of leukocytes adhering to a standardarea oriented perpendicular to the blood flow 1 mm apart from thebeginning of the coverslip. At a shear rate of 65/s and 280/s the areaon which the number of leukocytes was counted comprised 3.1 mm² and 2.1mm², respectively.

Results:

The P-selectin HuMabs inhibited the adhesion of leukocytes to theplatelet monolayer in a concentration-dependent manner. At a shear rateof 65/s and a concentration of 10 μg/ml the HuMabs inhibited theadhesion of leukocytes by 60-99%, preferably 70-99 %. The inhibitoryeffect of the HuMabs was more pronounced at the higher shear rate of280/s (closer to the arterial situation) as compared to the venous shearrate of 65/s. Overall, at a shear rate of 280/s the number of adheringleukocytes was lower than at 65/s. When comparing the Fc variants withthe respective parent antibodies, they had similar inhibitory activityin the ex vivo perfusion chamber, as demonstrated for HuMab 002 and itsvariants IgG4v1 and IgG1v1 (FIG. 5). The increased inhibitory activityof the mutants vs the parent antibody found in the in vitro assays wasnot observed in the ex vivo perfusion chamber which may be due to thesaturation of the Fcγ receptors of the leukocytes in whole human blood.

Effect of P-Selectin HuMabs on Leukocyte Adhesion to Endothelial Cells

Materials and Methods:

To address the anti-inflammatory potential of the P-selectin HuMabsunder shear conditions, the above-mentioned human blood flow system wasused in a set up in which endothelial cells were coated onto thecoverslips. Human umbilical vein endothelial cells (HUVEC) fromumbilical cords were isolated by digestion with collagenase Type II(Roche Switzerland) according to the method of Jaffe et al, 1993 (Pleaseadd full citation). They were cultivated in 1% gelatine-coated tissueculture flasks in medium 199 (M199, Sigma, Germany) supplemented with20% fetal calf serum (Gibco, Auckland), 100 IU/ml penicillin (Gibco,Auckland), 0.1 mg/ml streptomycin (Gibco, Auckland), 2 mmol/IL-glutamine (Gibco, Auckland), 10 U/ml heparin (Sigma) and 50 μg/ml ECgrowth supplement (Sigma, Germany). HUVECs were grown to confluency(approx. 4 days), passaged with trypsin/ethylendiaminetetraacetic acid(Gibco, Auckland) and seeded onto Thermanox plastic coverslips (approx200,000 ECs/coverslip) previously coated with 1% gelatine (Fluka,Germany). The HUVECs were allowed to settle and became confluent over1-2 days. They were stimulated with 20 ng/ml IL-4 (R&D Systems) 24 hbefore starting the perfusion and with 10⁻⁴ M histamine (Fluka, Germany)5-10 min prior to the perfusion. Each experiment was performed withHUVECs at passage 1. The coverslips with confluent monolayers ofstimulated HUVECs were positioned into the parallel plate perfusionchambers as described above. Similar to the perfusion experimentsdescribed above, whole blood was drawn from healthy donors. However inthese experiments, the blood was anticoagulated with a thrombininhibitor Ro-46-6240 (10 μM) and preincubated with differentconcentrations of the P-selectin antibodies (HuMabs, mutants, respectivereference antibodies) or human IgG1 and IgG4 as controls for 5 min justprior to the perfusion over the activated endothelial cells. The bloodflow was adjusted to 1 ml/min, the shear rate 65/s and the perfusiontime 5.5 min. After a washing period of 3 min with PBS, the HUVECs withthe adhering leukocytes were fixed with 3% paraformaldehyde for 2 minunder the same flow conditions as described. Then the coverslips wereremoved from the chambers, immersed in fresh fixative for 1 h, andstored in PBS-0.02% sodium azide. For morphometric analysis, theleukocytes were stained with a mouse antibody against the leukocytecommon antigen CD45, which was labeled beforehand using a modifiedbiotinylated anti-mouse immunoglobulin (Animal Research Kit, Dako, USA).The nuclei were counterstained with hematoxylin (J. T Baker, Holland).

Results:

The stimulation of the HUVECs with the combination of IL-4 and histamineresulted in the expression of P-selectin and the adhesion of differenttypes of leukocytes with granulocytes (including PMNs and eosinophils)constituting the prevailing portion of adhering leukocytes. The HuMabsof the invention inhibited the adhesion of the total leukocytepopulation by 60-90% at 3 μg/ml. Overall the inhibitory activity of theFc variants was not significantly different from that of the non-mutatedHuMabs.

The P-selectin HuMabs demonstrate a differential effect on the differentleukocyte subtypes. The effect on granulocytes is more pronounced ascompared to mononuclear leukocytes. The antibodies according to theinvention inhibited the adhesion of granulocytes (including PMNs andeosinophils) by 90-99%, monocytes by 50-88%, and lymphocytes by 5-40%.The respective decrease in the absolute numbers of the differentleukocyte subtypes is representatively given for IgG4v1 in FIG. 6.

Potential of P-Selectin HuMabs to Activate Complement System

C1q and C3c Binding ELISA:

To determine the ability of the antibodies of the invention to induceC1q binding and C3 activation, an ELISA approach was used. C1q is partof the adaptive immune system and, upon binding to immune complexes,triggers the sequential activation of several zymogens. The enzymes inturn, cause the cleavage of C3 molecules, which can result in the onsetof inflammatory reactions, opsonization of foreign or aberrant particlesand lysis of cell membranes.

In principle, the ELISA plate is coated with concentration ranges of theantibody, to which human C1q or human pooled serum, as a source of C3,is added. C1q or C3ε binding is detected by an antibody directed againsthuman C1q or C3ε followed by a peroxidase-labeled conjugate.

HuMab 002 (the hybridoma- and the transient transfectoma-derivedmaterial, its mutant variants, and control antibodies were tested inconcentrations of 0.16-20 μg/ml. As a negative control a human IgG4(CLB, the Netherlands, 0.5 μg/ml stock), that binds C1q very weakly, wasused. Human IgG1 (Sigma, 2 ug/ml stock) was incorporated as positivecontrol. For the detection of C1q, a rabbit antibody directed againstC1q (Dako) and a swine anti-rabbit IgG antibody, conjugated withhorseradish peroxidase (Sigma) were used. For the detection of C3ε amouse anti-human C3 antibody and a rabbit anti-mouse IgG antibody,conjugated with horseradish peroxidase (Sigma) were applied.

Calculations concerning EC50 values or maximum binding at 10 μg/ml(Bmax) of the HuMab tested were determined using nonlinear regressioncurve fitting (one site binding) using Graphpad Prism software.

Results:

HuMab 002 according to the invention was able to bind C1q efficiently asindicated by EC50 values of 0.946 μg/ml and 1.159 μg/ml, and Bmax(OD405) values of 0.987 and 0.711 for the hybridoma- andtransfectoma-derived material, respectively. As expected, the negativecontrol human IgG4 did not bind C1q, as indicated by a Bmax value of0.222 at OD405. However, all three Fc-variants tested (IgG4v1, IgG1v1,IgG1v2) had lost the capacity to bind C1q, as shown by OD405 Bmax valuesof 0.132, 0.119, and 0.132, respectively (Table 3). In line with the C1qbinding capacities, C3 deposition to HuMab 002 (hybridoma- andtransfectoma-derived) occurred in an antibody-concentration dependentmanner, and EC50 values ranged between 2.7 μg/ml and 8.3 μg/ml. However,all three Fc-variants were unable to initiate C3 deposition, asindicated by OD405 Bmax values of 0.104, 0.156 and 0.133, respectively(Table 3).

As HuMab 002 interacts with complement components, this antibody has theintrinsic potential to induce CDC in vivo. Therefore, the Fc part ofthis antibody is modified according to the invention. TABLE 3 Clq ELISAC3 ELISA Bmax Bmax (OD405 at Background (OD405 at Background 10 μg/ml)(OD405) 10 μg/ml) (OD405) HuMab 002 0.987 0.079 4.47 0.098 (hybridoma)IgG4v1 0.132 0.104 IgG1v1 0.0119 0.156 IgG1v2 0.132 0.133 HuMab 0020.711 4.071 (transient) IgG4 0.222 0.182

Potential of P-Selectin HuMabs to Bind to Fcγ Receptors

IgG antibody dependent cytotoxicity effects are mediated by Fcγreceptors on effector cells. Binding of hybridoma- andtransfectoma-derived HuMab 002 as well as the mutant variants andcontrol antibodies to FcγR expressing effector cells from human bloodwas studied by FACS analysis.

Materials and Methods:

FcγRI IIA1.6 transfectants or freshly isolated effector cells wereincubated with antibodies, and binding of antibody was detected withFITC-labeled rabbit-anti-human IgG F(ab)₂ (DAKO), or FITC-labeledrabbit-anti-human IgG F(ab)₂ (BD/Pharmingen). HuMab 002 (transienttransfectoma- and/or hybridoma-derived material, and mutant variants)wase tested at a concentration of 1 μg/ml (IIA1.6 transfectants) or 10μg/ml (effector cells). Absence of primary antibody or human IgG4 (10μg/ml) was used as negative control. To detect FcγRI expression onIIA1.6 cells, FITC-labeled mouse anti-human CD64 (BD/Pharmingen) wasused. In experiments using NK cell-enriched peripheral blood mononuclearcells, NK cells were identified by double staining using PE-labeledmouse-anti-human CD56 (BD/Pharmingen). Granulocytes and monocytes wereidentified based on FSC/SSC profile.

IIA1.6 cells, IIA1.6-FcγRI transfectant and freshly isolated effectorcells were incubated with antibodies. Binding of antibody was detectedwith FITC-labeled Rb-α-huIgG F(ab)₂ (DAKO), or FITC-labeled Rb-α-huIgGF(ab)₂ (BD/Pharmingen).

HuMab 002 (transient transfectoma-, hybridoma derived- and mutantvariant material) was tested at a concentration of 1 μg/ml in theIIA1.6-FcγRI transfectant binding assay. The IIA1.6 wild type cells wereused as a negative control. As a control for FcγRI expressionm-α-huCD64-FITC (BD/Pharmingen) was used.

HuMab 002 (transient transfectoma-, hybridoma derived- and mutantvariant material) was tested at a concentration of 10 μg/ml in theeffector cell binding assays. Transient transfectoma material was nottested in the granulocyte binding assay. IgG4 (10 μg/ml) was used as anegative control in all effector cell binding assays with the exceptionof the granulocyte binding assay.

Whole blood was enriched for NK cells using an NK isolation kit (DynalBiotech ASA, Oslo, Norway). NK cells were identified by m-α-huCD56-FITCstaining.

PBMCs (peripheral blood mononuclear cells) were obtained from wholeblood using Ficoll procedure as described in the protocol enclosed withthe NK isolation kit (Dynal Biotech ASA, Oslo, Norway). Monocytes wereidentified based on FSC/SSC profile.Granulocytes were isolated fromwhole blood using FACS lysis buffer and identified based on FSC/SSCprofile.

Freshly isolated effector cells were incubated with antibodies, andbinding of antibody was detected with FITC-labeled rabbit-anti-human IgGF(ab)₂ (DAKO), or FITC-labeled rabbit-anti-human IgG F(ab)₂(BD/Pharmingen). HuMab 002 (transient transfectoma- and/orhybridoma-derived material, and mutant variants) were tested at aconcentration of 10 μg/ml. Absence of primary antibody or human IgG4 (10μg/ml) was used as negative control. NK cells were isolated from MNCsamples by a NK isolation kit (Miltenyi Biotec, USA). In experimentsusing NK cell-enriched peripheral blood mononuclear cells, NK cells wereidentified by double staining using PE-labeled mouse-anti-human CD56(BD/Pharmingen). Granulocytes and monocytes were isolated according tothe state of the art from PBMC (e.g. Monocyte isolation kit (Miltenyi,see above). Granulocytes and monocytes were identified based on FSC/SSCprofile.

Results:

HuMab 002 according to the invention was able to bind to FcR asindicated by binding to granulocytes, monocytes and NK cells. All threeFc-variants tested (IgG4v1, IgG1v1 and IgG1v2) had completely lost thecapacity to bind to NK cells (Table 4). In addition, HuMab 002 boundefficiently to granulocytes and monocytes, whereas the mutant variantsshowed binding levels comparable to absence of primary antibody or humanIgG4, as indicated by percentages of cells binding antibody in Tables 5and 6. This indicates that the mutant variants lost the capacity tointeract with FcR on effector cells.

As HuMab 002 can efficiently interact with FcR, this antibody has theintrinsic potential to induce antibody dependent cell-mediatedcytotoxicity in vivo. Inactivation of the interaction with FcR asperformed for the Fc-variants according to the invention prevents ADCCin an effective manner. TABLE 4 NK cell binding Antibody (% NK cellsbinding antibody) No antibody 0.03 HuMab 002 (hybridoma) 90.92 HuMab 002(transient) 37.40 Human IgG4 0.06 IgG4v1 0.06 IgG1v1 0.12 IgG1v2 0.00

TABLE 5 Monocyte binding Antibody (% monocytes binding antibody) Noantibody 8.5 HuMab 002 (hybridoma) 38.4 HuMab 002 (transient) 31.3 HumanIgG4 9.4 IgG4v1 14.5 IgG1v1 12.3 IgG1v2 14.0

TABLE 6 Granulocyte binding (% granulocytes Antibody binding antibody)No antibody 1.2 HuMab 002 (hybridoma) 63.6 IgG4v1 1.6 IgG1v1 2.1 IgG1v22.0

1. An antibody containing a Fc part derived from human origin whereinthe antibody binds to P-selectin and is non-binding to complement factorC1q, said antibody being selected from the group consisting of (a) humansubclass IgG1 antibody comprising at least one mutation in L234, L235,D270, N297,E318, K320, K322, P331 and P329, and (b) human subclass IgG4antibody wherein S228 is replaced by P and L235 is replaced by E.
 2. Ananti-P-selectin antibody wherein the antibody is a human antibody whichbinds at least 1000 fold more specifically to P-selectin than to E- orL-selectin as measured by EC50 values in an Elisa assay, wherein the P—,E- and L-selectin are coated onto the microtiter plate.
 3. Ananti-P-selectin antibody wherein the antibody is a human antibody whichbinds at least 1000 fold more specifically to P-selectin than to E- orL-selectin as measured by EC50 values in an Elisa assay, wherein the P-and E- or L-selectin are coated onto the microtiter plate.
 4. A nucleicacid molecule encoding the antibody of claim 1, wherein the antibody isan antibody molecule.
 5. A vector comprising the nucleic acid moleculeof claim
 4. 6. A host cell comprising the vector of claim
 5. 7. Ananti-P-selectin antibody produced by a hybridoma cell line selected fromthe group consisting of DSM ACC2640, DSM ACC2641 and DSM ACC2642.
 8. Anucleic acid molecule encoding the antibody of claim 7, wherein theantibody is an antibody molecule.
 9. A vector comprising the nucleicacid molecule of claim
 8. 10. A host cell comprising the vector of claim9.
 11. A method for the preparation of the antibody of claim 1comprising the host cell of claim 6 under conditions that allowsynthesis and recovery of the antibody.
 12. A method for the preparationof the antibody of claim 7 comprising the host cell of claim 10 underconditions that allow synthesis and recovery of the antibody.
 13. Apharmaceutical composition comprising the antibody of claim
 1. 14. Thepharmaceutical composition of claim 13, further comprising at least onepharmaceutically acceptable excipient.
 15. A pharmaceutical compositioncomprising the antibody of claim
 7. 16. The pharmaceutical compositionof claim 15, further comprising at least one pharmaceutically acceptableexcipient.
 17. A method for treatment of a patient in need of therapycomprising administering to the patient a therapeutically effectiveamount of the antibody of claim
 1. 18. A medicament comprising theantibody of claim 1 for the treatment of inflammatory and thromboticdisorders.
 19. The medicament of claim 18 wherein the inflammatory andthrombotic disorders comprise PAOD or CLI.
 20. A kit to detect thepresence of P-selectin protein comprising the antibody of claim
 1. 21.The kit of claim 20 further comprising one or more of the groupconsisting the nucleic acid molecule of claim 4, the vector of claim 5and the host cell of claim 6.